Glucocorticoid Response Protein Research Articles

Absence of a secondary glucocorticoid response in C57BL/6J mice treated with topical dexamethasone.

Glucocorticoids such as dexamethasone can cause an increase in intraocular pressure (IOP) in some of the population, but not all. In this paper we used a mouse model of glucocorticoid induced ocular hypertension to examine the changes in the anterior segment of the eye in mice that failed to respond to glucocorticoid treatment with a sustained increase in IOP. C57BL/6J mice were treated with either 0.1% dexamethasone sodium phosphate ophthalmic solution or sterile PBS 3 times daily for up to 5 weeks. IOP was measured weekly at approximately the same time of the day. After 3–5 weeks of treatment, eyes were enucleated and evaluated for changes associated with steroid induced glaucoma. These studies showed that IOP was significantly elevated in dexamethasone (DEX) treated mice compared to PBS treated mice after 3 weeks of treatment, but IOP in DEX treated mice returned to baseline levels after 5 weeks of treatment. All the mice demonstrated a response to the glucocorticoid treatments and showed an elevation in FKBP5 expression after both 3 and 5 weeks of DEX treatment (primary glucocorticoid response protein) and a weight loss. Western blot analysis of anterior segments from treated mice, however, did not show an increase in secondary glucocorticoid response proteins such as β3 integrin or myocilin. Fibronectin levels were also not statistically different. The data suggest that in mice, which do not exhibit a prolonged increase in IOP in response to the DEX treatment, there is a compensatory mechanism that can prevent or turn off the secondary glucocorticoid response.

English

Myocilin (OMIM601652) MYOC gene, first revealed as a protein isolated from cultured human Trabecular Meshwork (TM) cells after prolong dexame thasone treatment. Hence given the name TIGR (Trabecular Meshwork Inducible Glucocorticoid Response Protein). The human myocilin gene, a concealed acidic glycoprotein,contains 504 amino acids, positioned at chromosome 1(1q24.3 ‐ 1q25.2), and filled with olfactomedin do main at its C terminus. Myocilin exists in various tissues throughout the eye and many other organs. M utant myocilin cause endoplasmic reticulum pressure in eye angle tissues, including the trabec ular meshwork, damaging the cells inside the meshwork, ultimately leading to structural changes in the outflow pathway, and high intraocular pressure.TM is vital in controlling pressure and mu tations in MYOC.It has been recognized as the cause of juvenile and adult onset of primary open a ngle glaucoma.

Thrombospondin-1 is a glucocorticoid responsive protein in humans.

Thrombospondin-1 (TSP1) is a matricellular protein whose gene expression has previously been shown to increase acutely after exposure to dexamethasone in vitro. The aim of this study was to determine if TSP1 is altered by acute and chronic states of glucocorticoid excess in human subjects. Three studies have been undertaken to assess the difference or change in TSP1 in response to altered glucocorticoid activity: i) an acute interventional study assessed the effects of a single 4 mg dose of dexamethasone in 20 healthy volunteers; ii) a cross-sectional study compared plasma TSP1 in 20 healthy volunteers and eight patients with Cushing's syndrome; iii) an interventional study assessed the effect on plasma TSP1 of an increase in hydrocortisone dose from ≤20 mg/day to 30 mg/day for 7 days in 16 patients with secondary adrenal insufficiency. In healthy volunteers, 4 mg dexamethasone significantly increased peripheral blood mononuclear cell (PBMC) TSP1 mRNA levels (P<0.0001) and plasma TSP1 concentrations (P<0.0001), peaking at 12 h. Median (interquartile range) plasma TSP1 was higher in Cushing's, 638 (535-756) ng/ml, than in healthy volunteers, 272 (237-336) ng/ml (P<0.0001). Plasma TSP1 >400 ng/ml diagnosed Cushing's syndrome with sensitivity of 100% and specificity of 85%. The higher hydrocortisone dose increased plasma TSP1 from 139 (86-199) to 256 (133-516) ng/ml, (P<0.01) in patients with secondary adrenal insufficiency. TSP1 is a glucocorticoid responsive protein in humans. Further research is required to determine if plasma TSP1 has a role as a glucocorticoid biomarker.

Functional characterization of a competitive peptide antagonist of p65 in human macrophage-like cells suggests therapeutic potential for chronic inflammation.

Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid responsive protein that links the nuclear factor-kappa B (NFκB) and the glucocorticoid signaling pathways. Functional and binding studies suggest that the proline-rich region at the carboxy terminus of GILZ binds the p65 subunit of NFκB and suppresses the immunoinflammatory response. A widely-used strategy in the discovery of peptide drugs involves exploitation of the complementary surfaces of naturally occurring binding partners. Previously, we observed that a synthetic peptide (GILZ-P) derived from the proline-rich region of GILZ bound activated p65 and ameliorated experimental encephalomyelitis. Here we characterize the secondary structure of GILZ-P by circular dichroic analysis. GILZ-P adopts an extended polyproline type II helical conformation consistent with the structural conformation commonly observed in interfaces of transient intermolecular interactions. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human macrophage-like THP-1 cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was nontoxic as determined by cell viability and apoptosis assays. Functionally, GILZ-P suppressed proliferation and glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, our data suggest that the GILZ-P has therapeutic potential in chronic CNS diseases where persistent inflammation leads to neurodegeneration such as multiple sclerosis and Alzheimer’s disease.

The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR.

Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma

PURPOSE: To screen a population with primary open-angle glaucoma for mutations in the gene that encodes the trabecular meshwork inducible glucocorticoid response protein (TIGR), also known as myocilin (MYOC). METHODS: Ophthalmologic information was collected for study subjects with primary open-angle glaucoma and their relatives. Mutation screening of 74 primary open-angle glaucoma probands was conducted by sequencing TIGR/MYOC coding sequence and splice sites. RESULTS: In 23 families we detected 13 nonsynonymous sequence changes, nine of which appear to be mutations likely to cause or contribute to primary open-angle glaucoma. Two mutations, Arg272Gly and Ile499Ser, and one nonsynonymous sequence variant, Asn57Asp, are novel. We found mutations in nine of 25 juvenile glaucoma probands (36%) and two of 49 adult-onset glaucoma probands (4%). Age classification of families rather than individual probands revealed mutations in three of nine families with strictly juvenile primary open-angle glaucoma (33%), and no mutations in 39 families with strictly adult-onset primary open-angle glaucoma (0%). In families with mixed-onset primary open-angle glaucoma containing both juvenile primary open-angle glaucoma and adult-onset primary open-angle glaucoma cases, we found mutations in eight of 26 families (31%). CONCLUSIONS: Our data suggest that Gly252Arg, Arg272Gly, Glu323Lys, Gln368STOP, Pro370Leu, Thr377Met, Val426Phe, Ile477Asn, and Ile499Ser are likely to play roles that cause or contribute to the etiology of autosomal dominant primary open-angle glaucoma. Our finding of more TIGR/MYOC mutations in families with mixed-onset primary open-angle glaucoma than in the families with strictly adult-onset primary open-angle glaucoma implies that the presence of relatives with juvenile primary open-angle glaucoma in a family could be used as a basis for identifying a subset of the population with adult-onset primary open-angle glaucoma with higher prevalence of TIGR/MYOC mutations. To address this issue, and to refine estimations of mutation prevalence in these age-defined subpopulations, prospective study of a larger population ascertained entirely through adult-onset primary open-angle glaucoma probands will be needed.

Immunohistochemical localization of MYOC/TIGR protein in the trabecular tissue of normal and glaucomatous eyes

Purpose. To examine immunohistochemically the localization of myocilin/trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) protein in the glaucomatous and normal trabecular meshworks. Methods. Trabecular tissues were used from one eye with late-onset goniodysgenetic glaucoma, three with primary open angle glaucoma (one of which had the MYOC/TIGR gene mutation), two with exfoliation glaucoma and one without glaucoma. For light microscopic immunohistochemistry, frozen sections were stained by the avidin-biotin complex method using anti-MYOC/TIGR polyclonal antibody. For electron microscopic immunohistochemistry, the pre-embedding method using the same antibody was performed. Double immunostaining using both anti-MYOC/TIGR and anti-type VI collagen antibodies was done by the immunofluorescence method. Results. With light microscopy, immunoreactivity was seen in the whole trabecular meshwork of each of the specimens. No notable differences were detected in staining among the types of glaucoma, or between the eyes with and those without the gene mutation. Under electron microscopy, immunoreaction products were observed not only in the cytoplasm of the trabecular cells but also in the extracellular matrix, where staining was associated with the long-spacing collagen, fine granular materials and possibly microfibrils. With double immunohistochemistry, MYOC/TIGR was colocalized with type VI collagen in the trabecular meshwork. Conclusions. In glaucomatous and normal trabecular meshworks, the MYOC/TIGR protein is distributed in the extracellular matrix colocalizing with type VI collagen.

The myocilin (MYOC) gene expression in the human trabecular meshwork

Purpose. We previously reported a novel cytoskeletal protein with a myosin-like domain which is localized in the ciliary rootlet and basal body of connecting cilium of photoreceptor and hence we named it ‘myocilin’. It was soon realized that myocilin is identical to a protein called TIGR (trabecular meshwork inducible glucocorticoid response protein) which was found to be responsible for the pathogenesis of juvenile open angle glaucoma. In this study, we employed in situ RNA hybridization to examine the myocilin (MYOC)/ TIGR gene expression in the trabecular meshworks of glaucomatous and nonglaucomatous eyes. Methods. The glaucomatous specimens were obtained by trabeculectomy from the patients with primary open angle glaucoma (POAG), chronic angle closure glaucoma (CACG) and steroid glaucoma, respectively, and the nonglaucomatous specimens were obtained from a victim of traffic accident at autopsy and from a patient with maxillary sinus carcinoma at enucleation for the operation. The in situ RNA hybridization was carried out with digoxigenin-labeled sense and antisense RNA probes. Results. In all cases, hybridization signals were detected primarily in the trabecular meshwork cells and secondarily in the fibroblast-like cells of corneoscleral wall. Conclusions. Myocilin gene is expressed clearly in the trabecular meshwork cells of both glaucomatous and nonglaucomatous eyes.

Detection of a New TIGR Gene Mutation in a Japanese Family With Primary Open Angle Glaucoma

Purpose: To describe a new mutation of the trabecular meshwork-inducible glucocorticoid response protein (TIGR) gene in a Japanese patient with familial primary open angle glaucoma (POAG). Methods: Standard ocular examinations were performed on the 44-year-old patient, his sister, and mother. DNA sequencing was used to identify the mutation. We also developed a DNA diagnostic method for detecting this missense mutation by polymerase chain reaction-induced mutation restriction analysis (PCR-IMRA). Results: The patient, father, and sister had been diagnosed as having POAG. The patient and his sister had a Thr448Pro mutation (C→A transition at the nucleotide number 1419) in exon 3. This mutation has not been reported before. Conclusions: Gene analysis is promising for an early diagnosis among the family members of familial POAG patients and will contribute to early therapy before an occurrence of irreversible visual impairment.

Immunofluorescence method for quantifying the trabecular meshwork glucocorticoid response (TIGR) protein in trabecular meshwork and Schlemm's canal cells

PURPOSE. Decreased flow of aqueous humor through the trabecular meshwork (TM) and into Schlemm's canal (SC) is believed to be a predominant factor in the development of the elevated intraocular pressure found in primary open-angle and steroid glaucoma. Recent biochemical and genetic evidence has suggested that alterations in the expression of the TM glucocorticoid response (TIGR) protein within the chamber angle may play a role in the development of these glaucomas. To understand the process of TIGR induction in outflow pathway cells we developed an assay for TIGR expression that could distinguish both individual cell responses and also provide a semi-quantitative comparison between cell cultures. The present study demonstrates this approach, using digital image capture of immunofluorescently stained human TM and SC cells after treatment with dexamethasone (Dex). METHODS. Confluent cultures of human TM and SC cells were treated with 1 M Dex or vehicle control for 10 days. The cells were then fixed, permeabilized, and stained using polyclonal antibodies produced against recombinant TIGR. Digital images of fluorescently stained cells, using the same exposure time within an experiment, were evaluated by tabulating the staining intensity of all cells on each image. Between 10 and 40 cells were evaluated per image, 8–10 frames/ sample, 2–3 samples per treatment. Each cell was ranked as either 0 (background), 1+ (minor), 2+ (moderate) or 3+ (very bright staining). RESULTS. TM cells showed a significant basal level of TIGR staining. About 20% of control cells showed appreciable levels of TIGR staining, with intensity levels evenly distributed between 1, 2 and 3+. Dex treatment increased the number of TM cells expressing TIGR to 60–80%, with the majority of cells showing 2+ to 3+ staining throughout the cytoplasm. SC cells showed no basal TIGR staining, but Dex-treated cells exhibited TIGR staining in 6–15% of cells. SC cell TIGR staining varied between 1+ to 2+ in intensity, and showed a distribution different from TM cells. Staining in SC cells was localized to a ribbon-like compartment adjacent to the nucleus. Such perinuclear localization was rarely seen in TM cells. CONCLUSIONS. The low standard errors of the mean TIGR responses within each experiment, and the reproducibility between experiments for each cell type, suggests good reliability for this method. At the same time, the consistent, marked contrast between TM and SC cells in their response to glucocorticoids demonstrates that the assay can distinguish significant differences between cell types. The data support the view that Dex has a cell type specific effect on TIGR induction. The different extent, pattern and localization of TIGR staining between cell types suggests that TIGR expression in SC cells could play a functional role in the outflow pathway, but one that may be distinct from that played in TM cells.

Novel TIGR/MYOC mutations in families with juvenile onset primary open angle glaucoma.

Mutations in the trabecular meshwork induced glucocorticoid response protein (TIGR) or myocilin (MYOC) has recently been shown to cause juvenile onset primary open angle glaucoma (JOAG). In this study, we...

Functional morphology of the trabecular meshwork in primate eyes.

The trabecular meshwork forms most of the resistance to aqueous humor outflow needed for maintenance of a pressure gradient between intraocular pressure of approximately 17 mmHg and venous pressure of approximately 10 mmHg. The composition of the extracellular material in the subendothelial or cribriform layer seems to be mainly responsible for outflow resistance. The aqueous humor pathways through the subendothelial layer can be influenced by ciliary muscle contraction and presumably also by contractile elements recently found both in trabecular meshwork and scleral spur. Pharmacologically induced disconnection of inner wall and cribriform cells leads to wash out of extracellular material through breaks of the endothelial lining of Schlemm's canal and to increase of outflow facility. In glaucomatous eyes the resistance to aqueous humor outflow is increased due to an increase in different forms of extracellular material deposited within the cribriform layer. The amount of this newly developed extracellular material is correlated with loss of axons in the optic nerve, indicating that a common factor is responsible for both changes. To investigate the effect of various factors on the biology of trabecular cells monolayer cultures derived from cribriform and corneoscleral trabecular meshwork have been established. The two cell lines can be differentiated because cribriform cells in vivo as in vitro stain for alphabeta-crystallin whereas the corneoscleral cells remain unstained. The effect of TGFbeta, a growth factor increased in aqueous humor of glaucomatous eyes and glycocorticoids on trabecular meshwork cells show typical changes in formation of extracellular matrix components and of stress proteins. Dexamethasone and oxidative damage also lead to increase of trabecular meshwork inducible glucocorticoid response (TIGR) protein. A mutation of the TIGR-gene family has recently been found in families with juvenile and chronic simple glaucoma. Future research has to clarify the significance of these genetic factors for the pathophysiology of glaucoma and the role of trabecular cell activity in this respect.

Novel mutations in the TIGR gene in early and late onset open angle glaucoma.

A gene for juvenile onset, open angle glaucoma (JOAG) has been localized to chromosome 1q21-31 in several families. Mutations in the trabecular meshwork-induced glucocorticoid response protein (TIGR) gene, which maps to this region, recently have been found in families segregating both JOAG and a later onset form of primary open angle glaucoma (POAG). We have analysed the TIGR gene in two families; one Spanish family segregating autosomal dominant JOAG and an Irish family with a later onset form of autosomal dominant POAG. We have found a G-T transversion in the first base of codon 426 in all affected members of the Spanish family, which results in a valine to phenylalanine amino acid substitution. We have also found a G-A transition at the first base of codon 367 that segregates through all but one branch of the Irish family and results in a glycine to arginine amino acid substitution. Members of this family that carry the Gly367Arg change also share a common haplotype that is neither present in any of the unaffected members of the family, nor in the branch that does not segregate the mutation. Identification of further mutations in the TIGR gene increases its importance in the etiology of open angle glaucoma.

Novel mutations in the TIGR gene in early and late onset open angle glaucoma

A gene for juvenile onset, open angle glaucoma (JOAG) has been localized to chromosome 1q21-31 in several families. Mutations in the trabecular meshwork-induced glucocorticoid response protein (TIGR) gene, which maps to this region, recently have been found in families segregating both JOAG and a later onset form of primary open angle glaucoma (POAG). We have analysed the TIGR gene in two families; one Spanish family segregating autosomal dominant JOAG and an Irish family with a later onset form of autosomal dominant POAG. We have found a G-T transversion in the first base of codon 426 in all affected members of the Spanish family, which results in a valine to phenylalanine amino acid substitution. We have also found a G-A transition at the first base of codon 367 that segregates through all but one branch of the Irish family and results in a glycine to arginine amino acid substitution. Members of this family that carry the Gly367Arg change also share a common haplotype that is neither present in any of the unaffected members of the family, nor in the branch that does not segregate the mutation. Identification of further mutations in the TIGR gene increases its importance in the etiology of open angle glaucoma. Hum Mutat 11:244–251, 1998. © 1998 Wiley-Liss, Inc.

Recent advances in molecular genetics of glaucomas.

Glaucomas are a heterogeneous group of eye conditions with manifestation from as early as birth to very late age of onset in life. The primary type of these conditions affecting children and juveniles are less frequent, but the prevalence of glaucomas affecting older people of > or = 70 years progressively rises to approximately 5%. The molecular genetics of three types of glaucoma have been the subject of investigation in the last few years. As a result, two loci (GLC3A and GLC3B) have been identified for primary congenital glaucoma, one locus (GLC1A) for juvenile-onset primary open angle glaucoma and a further two loci (GLC1B and GLC1C) for late-onset chronic open angle glaucoma. Early this year, the first set of mutations was described in the CYP1B1 (Cytochrome P4501B1) and TIGR (Trabecular meshwork Inducible Glucocorticoid Response Protein) genes for the GLC3A and GLC1A-linked families, respectively. The mapping of different types of glaucoma and mutation identification in these two genes are the focus of this review.

Repression of glucocorticoid receptor transactivation and DNA binding of a glucocorticoid response element within the serum/glucocorticoid-inducible protein kinase (sgk) gene promoter by the p53 tumor suppressor protein.

sgk is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated by serum and glucocorticoids in Rat2 fibroblasts and in mammary epithelial cells. 5'-Deletion analysis of the sgk promoter, using a series of sgk-CAT. (chloramphenicol acetyltransferase) chimeric reporter gene plasmids, defined a glucocorticoid-responsive region that contains a glucocorticoid response element (sgkGRE) between -1000 and -975 bp. The sgkGRE is specifically bound by glucocorticoid receptors and is sufficient to confer glucocorticoid responsiveness to a heterologous promoter in several cell lines. Strikingly, cotransfection of either the murine or human wild type p53, but not a mutant p53, repressed the dexamethasone-stimulated transactivation of reporter plasmids containing either the sgkGRE or a consensus GRE. Gel shift analysis revealed that in vitro synthesized p53 prevented binding of the glucocorticoid receptor both to the sgkGRE as well as to a consensus GRE. The p53-mediated repression of dexamethasone-induced sgkGRE activity required both the DNA binding and transactivation functions of the p53 protein. Activation of endogenous p53, by exposure to UV light, repressed the glucocorticoid receptor transactivation of a consensus GRE-CAT reporter plasmid in transfected cells. Conversely, activated glucocorticoid receptors suppressed the transactivation function of p53, while transrepression by p53 was largely unaffected. The presented data demonstrate that sgk is a primary glucocorticoid-responsive protein kinase gene that implicates a new pathway of cross-talk between steroid receptor signaling and cellular phosphorylation cascades. In addition, our study provides the first evidence of mutual interference of transactivation functions of p53 and the glucocorticoid receptor, possibly through their direct interaction.

Identification of a new ‘TIGR’ mutation in a family with juvenile-onset primary open angle glaucoma

Positional mapping of families segregating for juvenile-onset primary open angle glaucoma (JOAG) has previously identified a locus (GLC1A) for this condition on the long arm of chromosome 1. Recently, three mutations in the TIGR gene (Trabecular meshwork Inducible Glucocorticoid Response protein) have been described in a total of ten familial, three sporadic, and one normal subject. One of the familial cases has also been indicated to be of an adult-onset type. Herein, we report the identification of a new mutation in the TIGR gene in a six generation well-documented Edinburgh family with JOAG. We have sampled and screened the living affected members of this family and identified an ‘A’-to-‘G’ transition at amino acid 337 that has changed the glutamine (Gin) to arginine (Arg). This newly identified mutation resides 27 amino acids 5-prime from the previously reported mutation of Gly-to-Val. This mutation created a new MspI restriction site that has co-segregated perfectly with inherited affected haplotype of the pedigree and, furthermore, it has not been observed in 142 chromosomes of randomly selected subjects of the same population. This report, therefore, confirms the role of the TIGR gene in the etiology of JOAG and adds a new mutation to the three reported previously.

De novo synthesis of glucocorticoid hormone regulated inner ear proteins in rats

Changes of rat inner ear de novo protein synthesis in response to dexamethasone (DEX), a synthetic glucocorticoid, have been analyzed by high resolution two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-SDS-PAGE) and fluorography. Two proteins ( M r 41 000 and 35 000) were amplified and one protein ( M r 47 000) was suppressed by DEX in a cochlear culture medium. In the culture medium conditioned by vestibular tissue, three proteins ( M r 67 000, 57 000 and 50 000) were amplified after DEX administration. In cochlear and vestibular tissues, glucocorticoid-responsive protein synthesis was down-regulated by DEX, including two proteins ( M r 39 000 and 35 000) in the cochlea and five proteins ( M r 80 000, 64 000, 59 000, 56 000 and 40 000) in the vestibule. The regulation of these inner ear proteins by DEX suggests that glucocorticoid may play an important role in normal inner ear microhomeostasis, as well as in the treatment of some inner ear disorders.

Regulated endocrine-specific protein-18: a short-lived novel glucocorticoid-regulated endocrine protein

Regulated endocrine-specific protein-18 (RESP18) is an 18-kilodalton endocrine-specific transcript whose expression is regulated by a number of different physiological and pharmacological stimuli in different tissues. RESP18 messenger RNA was identified in all cell types in the anterior pituitary, at levels that varied 2-fold from the lowest (corticotropes and thyrotropes) to the highest (gonadotropes, somatotropes, and mammotropes); the melanotropes of the intermediate pituitary have levels of RESP18 messenger RNA comparable to the highest levels in cells in the anterior pituitary. Mouse RESP18 was cloned and used as the basis for biosynthetic studies on RESP18 in AtT-20 cells, which express RESP18 endogenously; mouse RESP18 was highly homologous to rat RESP18. Pulse-chase biosynthetic labeling studies showed that AtT-20 cells expressed much less RESP18 than the endogenous prohormone, POMC, but that glucocorticoid treatment lowered POMC and raised RESP18 biosynthetic rates so that they were nearly equimolar. Surprisingly, RESP18 was not processed to smaller peptides to any significant extent, nor was RESP18 or any smaller peptide secreted. Newly synthesized RESP18 normally disappeared from AtT-20 cell extracts with a half-life of less than 15 min; the intracellular half-life of RESP18 was increased strikingly after glucocorticoid treatment of the cells. Upon subcellular fractionation, RESP18 was found to be entirely particulate and to cofractionate with markers for the endoplasmic reticulum, rather than with markers for secretory granules, such as POMC and prohormone-processing enzymes. Therefore, RESP18 is a major glucocorticoid-responsive protein in the secretory pathway of corticotropes, but its function may be entirely within the neuroendocrine cell.

Regulated endocrine-specific protein-18: a short-lived novel glucocorticoid-regulated endocrine protein.

Regulated endocrine-specific protein-18 (RESP18) is an 18-kilodalton endocrine-specific transcript whose expression is regulated by a number of different physiological and pharmacological stimuli in different tissues. RESP18 messenger RNA was identified in all cell types in the anterior pituitary, at levels that varied 2-fold from the lowest (corticotropes and thyrotropes) to the highest (gonadotropes, somatotropes, and mammotropes); the melanotropes of the intermediate pituitary have levels of RESP18 messenger RNA comparable to the highest levels in cells in the anterior pituitary. Mouse RESP18 was cloned and used as the basis for biosynthetic studies on RESP18 in AtT-20 cells, which express RESP18 endogenously; mouse RESP18 was highly homologous to rat RESP18. Pulse-chase biosynthetic labeling studies showed that AtT-20 cells expressed much less RESP18 than the endogenous prohormone, POMC, but that glucocorticoid treatment lowered POMC and raised RESP18 biosynthetic rates so that they were nearly equimolar. Surprisingly, RESP18 was not processed to smaller peptides to any significant extent, nor was RESP18 or any smaller peptide secreted. Newly synthesized RESP18 normally disappeared from AtT-20 cell extracts with a half-life of less than 15 min; the intracellular half-life of RESP18 was increased strikingly after glucocorticoid treatment of the cells. Upon subcellular fractionation, RESP18 was found to be entirely particulate and to cofractionate with markers for the endoplasmic reticulum, rather than with markers for secretory granules, such as POMC and prohormone-processing enzymes. Therefore, RESP18 is a major glucocorticoid-responsive protein in the secretory pathway of corticotropes, but its function may be entirely within the neuroendocrine cell.