Review paper: origin and molecular pathology of adrenocortical neoplasms.

Neoplastic adrenocortical lesions are common in humans and several species of domestic animals. Although there are unanswered questions about the origin and evolution of adrenocortical neoplasms, analysis of human tumor specimens and animal models indicates that adrenocortical tumorigenesis involves both genetic and epigenetic alterations. Chromosomal changes accumulate during tumor progression, and aberrant telomere function is one of the key mechanisms underlying chromosome instability during this process. Epigenetic changes serve to expand the size of the uncommitted adrenal progenitor population, modulate their phenotypic plasticity (i.e., responsiveness to extracellular signals), and increase the likelihood of subsequent genetic alterations. Analyses of heritable and spontaneous types of human adrenocortical tumors documented alterations in either cell surface receptors or their downstream effectors that impact neoplastic transformation. Many of the mutations associated with benign human adrenocortical tumors result in dysregulated cyclic adenosine monophosphate signaling, whereas key factors and/or signaling pathways associated with adrenocortical carcinomas include dysregulated expression of the IGF2 gene cluster, activation of the Wnt/beta-catenin pathway, and inactivation of the p53 tumor suppressor. A better understanding of the factors and signaling pathways involved in adrenal tumorigenesis is necessary to develop targeted pharmacologic and genetic therapies.

GATA-4 is a granulosa cell factor employed in inhibin-alpha activation by the TGF-beta pathway.

Part of heterodimeric inhibin, inhibin-alpha is crucial for mammalian ovarian function. Regulation of inhibin-alpha expression in granulosa cells is both endocrine, primarily by follicle-stimulating hormone (FSH), and paracrine, primarily by members of the transforming growth factor beta (TGF-beta) superfamily. Smad proteins transmit TGF-beta signals to the nucleus, but the cooperating transcription factors involved in inhibin-alpha promoter activation remain unknown. Transcription factor GATA-4 regulates inhibin-alpha in gonadal cells, and the FSH cascade activates GATA-4. We hypothesized that the TGF-beta signalling cascade and GATA-4 also cooperate to regulate inhibin-alpha expression. In KK-1 granulosa tumour cells, which resemble normal granulosa cells and express inhibin-alpha, we found that TGF-beta upregulated GATA-4 expression. Transient transfection experiments in KK-1 cells demonstrated that dominant negative GATA-4 variants or mutations of GATA-binding sites in the inhibin-alpha promoter attenuated TGF-beta-induced gene activation. In GATA-4-deficient COS-7 cells, TGF-beta enhanced the expression of the inhibin-alpha promoter only in the presence of exogenous GATA-4. Smad3, but not Smad2, cooperated with GATA-4 in the transcriptional activation of the inhibin-alpha promoter, and immunoprecipitation experiments in KK-1 cells revealed a physical Smad3:GATA-4 interaction. Our data suggest that GATA-4, interacting with Smad3, is a cofactor for TGF-beta signalling to activate inhibin-alpha in granulosa cells.

Gonadectomy-induced adrenocortical neoplasia in the domestic ferret (Mustela putorius furo) and laboratory mouse.

Sex steroid-producing adrenocortical adenomas and carcinomas occur frequently in neutered ferrets, but the molecular events underlying tumor development are not well understood. Prepubertal gonadectomy elicits similar tumors in certain inbred or genetically engineered strains of mice, and these mouse models shed light on tumorigenesis in ferrets. In mice and ferrets, the neoplastic adrenocortical cells, which functionally resemble gonadal steroidogenic cells, arise from progenitors in the subcapsular or juxtamedullary region. Tumorigenesis in mice is influenced by the inherent susceptibility of adrenal tissue to gonadectomy-induced hormonal changes. The chronic elevation in circulating luteinizing hormone that follows ovariectomy or orchiectomy is a prerequisite for neoplastic transformation. Gonadectomy alters the plasma or local concentrations of steroid hormones and other factors that affect adrenocortical tumor development, including inhibins, activins, and Müllerian inhibiting substance. GATA-4 immunoreactivity is a hallmark of neoplastic transformation, and this transcription factor might serve to integrate intracellular signals evoked by different hormones. Synergistic interactions among GATA-4, steroidogenic factor-1, and other transcription factors enhance expression of inhibin-alpha and genes critical for ectopic sex steroid production, such as cytochrome P450 17alpha-hydroxylase/17,20 lyase and aromatase. Cases of human adrenocortical neoplasia have been linked to precocious expression of hormone receptors and to mutations that alter the activity of G-proteins or downstream effectors. Whether such genetic changes contribute to tissue susceptibility to neoplasia in neutered ferrets and mice awaits further study.

Transcription factor GATA-4 is a marker of anaplasia in adrenocortical neoplasms of the domestic ferret (Mustela putorius furo).

Adrenocortical neoplasms are a common cause of morbidity in neutered ferrets. Recently we showed that gonadectomized DBA/2J mice develop adrenocortical tumors that express transcription factor GATA-4. Therefore, we screened archival specimens of adrenocortical neoplasms from neutered ferrets to determine whether GATA-4 could be used as a tumor marker in this species. Nuclear immunoreactivity for GATA-4 was evident in 19/22 (86%) of ferret adrenocortical carcinomas and was prominent in areas exhibiting myxoid differentiation. Normal adrenocortical cells lacked GATA-4 expression. Two other markers of adrenocortical tumors in gonadectomized mice, inhibin-alpha and luteinizing hormone receptor, were coexpressed with GATA-4 in some of the ferret tumors. No GATA-4 expression was observed in three cases of nodular hyperplasia, but patches of anaplastic cells expressing GATA-4 were evident in 7/14 (50%) of tumors classified as adenomas. We conclude that GATA-4 can function as a marker of anaplasia in ferret adrenocortical tumors.

Nude mice as a model for gonadotropin-induced adrenocortical neoplasia.

Certain inbred mice (e.g., DBA/2J, CE) develop sex steroid producing adrenocortical tumors following gonadectomy. This adrenal response is thought to result from an unopposed increase in circulating gonadotropins and/or a decrease in factor(s) of gonadal origin. To differentiate between these two possibilities, we utilized the NU/J strain of nude mice, which are immunologically compromised and therefore permissive to xenografts. One group of female nude mice was gonadectomized, while another group of females received xenografts of CHO cells stably transfected with human chorionic gonadotropin (hCG). After 1-2 months, subcapsular adrenocortical neoplasms containing sex steroid-producing cells were observed in both groups. We conclude that high levels of circulating gonadotropins are sufficient to induce adrenocortical tumorigenesis, even in the presence of intact gonads.

Reciprocal changes in the expression of transcription factors GATA-4 and GATA-6 accompany adrenocortical tumorigenesis in mice and humans.

While certain genetic changes are frequently found in adrenocortical carcinoma cells, the molecular basis of adrenocortical tumorigenesis remains poorly understood. Given that the transcription factors GATA-4 and GATA-6 have been implicated in gene expression and cellular differentiation in a variety of tissues, including endocrine organs such as testis, we have now examined their expression in the developing adrenal gland, as well as in adrenocortical cell lines and tumors from mice and humans. Northern blot analysis and in situ hybridization revealed abundant GATA-6 mRNA in the fetal and postnatal adrenal cortex of the mouse. In contrast, little or no GATA-4 expression was detected in adrenal tissue during normal development. In vivo stimulation with ACTH or suppression with dexamethasone did not affect the expression of GATA-4 or GATA-6 in the murine adrenal gland. To assess whether changes in the expression of GATA-4 or GATA-6 accompany adrenocortical tumorigenesis, we employed an established mouse model. When gonadectomized, inhibin alpha/SV40 T-antigen transgenic mice develop adrenocortical tumors in a gonadotropin-dependent fashion. In striking contrast to the normal adrenal glands, GATA-6 mRNA was absent from adrenocortical tumors or tumor-derived cell lines, while GATA-4 mRNA and protein were abundantly expressed in the tumors and tumor cell lines. Analogous results were obtained with human tissue samples; GATA-4 expression was detected in human adrenocortical carcinomas but not in normal tissue, adenomas, or pheochromocytomas. Taken together these results suggest different roles for GATA-4 and GATA-6 in the adrenal gland, and implicate GATA-4 in adrenal tumorigenesis. Immunohistochemical detection of GATA-4 may serve as a useful marker in the differential diagnosis of human adrenal tumors.

Distinct roles for visceral endoderm during embryonic mouse development.

The murine visceral endoderm is an extraembryonic cell layer that appears prior to gastrulation and performs critical functions during embryogenesis. The traditional role ascribed to the visceral endoderm entails nutrient uptake and transport. Besides synthesizing a number of specialized proteins that facilitate uptake, digestion, and secretion of nutrients, the extraembryonic visceral endoderm coordinates blood cell differentiation and vessel formation in the adjoining mesoderm, thereby facilitating efficient exchange of nutrients and gases between the mother and embryo. Recent studies suggest that in addition to this nutrient exchange function the visceral endoderm overlying the egg cylinder stage embryo plays an active role in guiding early development. Cells in the anterior visceral endoderm function as an early organizer. Prior to formation of the primitive streak, these cells express specific gene products that specify the fate of underlying embryonic tissues. In this review we highlight recent investigations demonstrating this dual role for visceral endoderm as a provider of both nutrients and developmental cues for the early embryo.

Expression and regulation of transcription factors GATA-4 and GATA-6 in developing mouse testis.

Previous studies have shown that transcription factors GATA-4 and GATA-6 are expressed in granulosa and thecal cells of the mouse ovary and that GATA-4 expression in ovarian tissue is regulated by gonadotropins. Given the emerging role of GATA-4 and GATA-6 in gonadal cells, we have now studied the expression and regulation of these factors in the mouse testis and testicular cell lines. In situ hybridization demonstrated GATA-4 messenger RNA (mRNA) in the fetal testis at 13.5 days postcoitum. Both GATA-4 and GATA-6 transcripts were observed in late fetal, neonatal, juvenile, and adult Sertoli cells. In addition, GATA-4 mRNA was detected in interstitial cells throughout development. Immunohistochemistry demonstrated GATA-4 protein in both Sertoli and Leydig cells in postnatal animals. The regulation of GATA-4 and GATA-6 expression was explored using established testicular cell lines. Treatment of Leydig tumor cell lines with hCG resulted in a modest, but statistically significant, increase in the steady state level of GATA-4 mRNA, comparable to the previously described effect of FSH on GATA-4 expression in Sertoli cell lines. Gonadotropin or androgen action was not, however, a prerequisite for the basal expression of GATA-4 or GATA-6 in the testis, as their presence in Sertoli and Leydig cells was demonstrated in genetically hypogonadal hpg mice, in rats treated with GnRH receptor antagonist, and in Sertoli cells after chemical abolition of Leydig cells. Cotransfection studies using a GATA-4 expression plasmid and an inhibin alpha promoter/reporter gene construct in Leydig and granulosa tumor cell lines revealed that the inhibin alpha promoter harboring essential GATA-binding sites can be trans-activated by GATA-4. In light of these results, we propose that transcription factors GATA-4 and GATA-6 play differing roles in the maturation and function of testicular somatic cells.

[Own experience in the application of midazolam (Dormicum) before the diagnostic examinations an short surgical procedures in children]. / Doswiadczenia wlasne w zastosowaniu midazolamu (Dormicum) przed badaniami diagnostycznymi i krótkimi zabiegami chirurgicznymi u dzieci.

In this paper the evaluation of midazolam (Dormicum) efficiency in obtaining basic sedative effect in 36 children during diagnostic examinations and short surgical operations was done. In 26 patients good sedative effect was obtained. Forgetfulness was present in 17 from 25 patients above 2 years of life, and partial in the rest 8 patients. Fear reactions were observed in 10 children with lower doses of this medicine. Side effects were observed in 8 patients, and had a transient character and were not dangerous for the patients' life.

[The lack of parental acceptance of the stress associated with neoplastic disease of the child as a cause of treatment failure]. / Brak akceptacji rodziców wobec stresu zwiazanego z choroba nowotworowa dziecka jako przyczyna zaniechania leczenia.

Three unsuccessful cases of neoplastic diseases in children were described. No parents cooperation with treatment center led after good initial response to the progression of disease and the death of children. The reason of such parents' decisions was incapability to manage the stress caused by child's disease.

Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells.

In situ hybridization studies, promoter analyses and antisense RNA experiments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In this study, we utilized Gata4-/- embryonic stem (ES) cells to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. First, we assessed the ability of Gata4-/- ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes were seen in both wild-type and Gata4-/- embryoid bodies, although cardiomyocytes were observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4-/- embryoid bodies revealed the presence of sarcomeres and junctional complexes, while immunofluorescence confirmed the presence of cardiac myosin. To assess the capacity of Gata4-/- ES cells to differentiate into cardiomyocytes in vivo, we prepared and analyzed chimeric mice. Gata4-/- ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4-/- ES cells contributed to endocardium, myocardium and epicardium. In situ hybridization showed that myocardium derived from Gata4-/- ES cells expressed several cardiac-specific transcripts, including cardiac alpha-myosin heavy chain, troponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6. Taken together these results indicate that GATA-4 is not essential for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4.

Wild-type endoderm abrogates the ventral developmental defects associated with GATA-4 deficiency in the mouse.

GATA-4 knockout mice die by 9.5 days postcoitum and exhibit profound defects in ventral morphogenesis, including abnormal foregut formation and a failure of fusion of the bilateral myocardial primordia. During early mouse development, GATA-4 is expressed in cardiogenic splanchnic mesoderm and associated endoderm, suggesting that the presence of this transcription factor in one or both of these tissue types is essential for ventral development. To distinguish whether GATA-4 expression in mesoderm or endoderm accounts for the phenotype of the knockout mouse, we prepared chimeric mice by injecting Gata4-/- ES cells into 8-cell stage ROSA26(Gata4+/+) embryos. We identified a series of high percentage null chimeras (8-10 days postcoitum) in which Gata4+/+ cells were restricted to visceral yolk sac endoderm and small portions of the foregut/hindgut endoderm. Despite an absence of GATA-4 in all other cells of these embryos, there was normal development of the heart, foregut, and surrounding tissues. We conclude that expression of GATA-4 in endoderm rather than cardiogenic mesoderm is required for ventral morphogenesis.

Expression and hormonal regulation of transcription factors GATA-4 and GATA-6 in the mouse ovary.

Two members of the GATA-binding family of transcription factors, GATA-4 and GATA-6, are expressed in the vertebrate ovary. To gain insight into the role of these factors in ovarian cell differentiation and function, we used in situ hybridization to determine the patterns of expression of GATA-4 and GATA-6 in mouse ovary during development and in response to hormonal stimulation. GATA-4 messenger RNA (mRNA) was first evident in the ovary around the time of birth. In the adult ovary, abundant GATA-4 mRNA was detected in granulosa cells of primary and antral follicles, with lesser amounts of GATA-4 message detected in theca cells, germinal epithelium, and interstitial cells. Little or no GATA-4 mRNA was found in corpus luteum. GATA-6 message exhibited a different distribution in the ovary, with abundant expression evident in both granulosa cells and corpora lutea. Stimulation of 3-week-old females with PMSG or estrogen enhanced follicular expression of GATA-4 and GATA-6 transcripts. Subsequent induction of ovulation with human CG resulted in a decrease in GATA-4 mRNA expression in granulosa cells, whereas GATA-6 mRNA expression persisted in granulosa cells after ovulation and in corpora lutea. Moreover, follicular apoptosis was associated with a decrease in the expression of GATA-4 but not GATA-6 message. Stimulation of cultured gonadal cell lines with FSH resulted in increased expression of GATA-4 message, whereas GATA-6 mRNA expression was not affected. In light of these findings, the established role of other GATA-binding proteins in hematopoetic cell differentiation and apoptosis, and the presence of conserved GATA motifs in the promoters of genes expressed selectively in ovary, we propose that GATA-4 and GATA-6 play distinct roles in follicular development and luteinization.

Induction of yolk sac endoderm in GATA-4-deficient embryoid bodies by retinoic acid.

GATA-4, a transcription factor implicated in lineage determination, is expressed in both parietal and visceral endoderm of the early mouse embryo. In embryonic stem cell-derived embryoid bodies, GATA-4 mRNA is first detectable at 4-5 days of differentiation and is confined to visceral endoderm cells on the surface of the bodies. Previously we reported that targeted mutagenesis of the Gata4 gene in embryonic stem cells results in a block in visceral endoderm differentiation in vitro. In an attempt to elucidate the role of GATA-4 in the formation of visceral endoderm, we have now differentiated Gata4 -/- and wild type embryoid bodies in the presence of retinoic acid +/- dbcAMP, known inducers of endoderm formation. We show that differentiation of Gata4 -/- embryoid bodies in the presence of retinoic acid results in formation of visceral endoderm, while differentiation of Gata4 -/- embryoid bodies in the presence of retinoic acid plus dbcAMP causes parietal endoderm formation. The presence of these yolk sac endoderm layers was confirmed by light microscopy and analysis of biochemical markers including alpha-fetoprotein, type IV collagen, laminin, and binding sites for Dolichos biflorus agglutinin. Treatment of Gata4 -/- embryoid bodies with retinoic acid induces expression of another GATA-binding protein, GATA-6, in both visceral and parietal endoderm cells. That another GATA-binding protein is induced in the absence of GATA-4 suggests that this family of transcription factors plays an important role in yolk sac differentiation.

Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm.

During mouse embryogenesis the first hematopoietic and endothelial cells form in blood islands located between layers of visceral endoderm and mesoderm in the yolk sac. The role of visceral endoderm in primitive hematopoiesis and vasculogenesis is not well understood. We have assessed the consequences of a lack of visceral endoderm on blood cell and vessel formation using embryoid bodies derived from mouse embryonic stem (ES) cells deficient in GATA-4, a transcription factor expressed in yolk sac endoderm. When differentiated in vitro, these mutant embryoid bodies do not develop an external visceral endoderm layer. We found that Gata4-/- embryoid bodies, grown either in suspension culture or attached to a substratum, are defective in primitive hematopoiesis and vasculogenesis as evidenced by a lack of recognizable blood islands and vascular channels and a reduction in the expression of the primitive erythrocyte marker epsilon y-globin. Expression of the endothelial cell transcripts FIk-1, FIt-1, and platelet-endothelial cell adhesion molecule (PECAM) was not affected in the mutant embryoid bodies. Gata4-/- ES cells retained the capacity to differentiate into primitive erythroblasts and endothelial cells when cultured in methylcellulose or matrigel. Analysis of chimeric mice, generated by injecting Gata4-/- ES cells into 8-cell stage embryos of ROSA26 transgenic animals, showed that Gata4-/- ES cells can form blood islands and vessels when juxtaposed to visceral endoderm in vivo. We conclude that the visceral endoderm is not essential for the differentiation of primitive erythrocytes or endothelial cells, but this cell layer plays an important role in the formation and organization of yolk sac blood islands and vessels.

The gene for transcription factor GATA-6 resides on mouse chromosome 18 and is expressed in myocardium and vascular smooth muscle.

We report the mapping and developmental expression pattern of the gene encoding mouse GATA-6, a member of a family of transcription factors involved in tissue-specific gene expression. Using backcross analysis, the Gata6 gene was localized to mouse chromosome 18, linked to the gene encoding transthyretin. RNase protection analysis showed that Gata6 is abundantly expressed in the heart, stomach, intestine, and ovaries of the adult mouse. The developmental expression patterns of Gata6 and the closely related gene Gata4 were directly compared using in situ hybridization. Both genes were found to be highly expressed in the myocardium, stomach epithelium, and small intestinal epithelium throughout mouse development. Of the two genes, however, only Gata6 was expressed in vascular smooth muscle. The overlapping distributions of GATA-4 and GATA-6 transcripts in the heart support the possibility of functional redundancy or interplay between these two transcription factors in this tissue. The presence of GATA-6 mRNA in vascular smooth muscle suggests that this transcription factor may play a distinctive role in gene expression in this cell type.

Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro.

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata-4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.

Regulation of J6 gene expression by transcription factor GATA-4.

Retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells into primitive endoderm is accompanied by increased transcription of the gene for J6, a heat shock protein implicated in collagen biosynthesis. In this paper we present evidence that transcription factor GATA-4, a retinoic acid-inducible GATA-binding protein, is involved in the regulation of J6 gene expression in F9 cells. Northern-blot analysis indicates that transcripts encoding GATA-4 and J6 increase in parallel during retinoic acid-induced differentiation of F9 cells. Gel-shift experiments and antibody binding studies demonstrate that: (1) GATA-4 is the major GATA-binding protein activity in differentiated F9 cells, and (2) GATA-4 binds to consensus GATA motifs in the retinoic acid-responsive portion of the J6 promoter. Co-transfection studies using NIH 3T3 cells show that GATA-4 is a potent trans-activator of the J6 promoter. These lines of evidence suggest that expression of J6 in F9 cells is regulated by GATA-4. We speculate that transcription factor GATA-4 may also control other genes involved in extracellular matrix formation in the yolk sac.

Mutagenesis of cysteine residues in the human gonadotropin alpha subunit. Roles of individual disulfide bonds in secretion, assembly, and biologic activity.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha subunit but differ in their hormone-specific beta subunits. Site-directed mutagenesis was used to examine the role of the five disulfide bonds in the alpha subunit on the folding, assembly with the hCG beta subunit, and in cases where dimer formation occurred, receptor binding and signal transduction. Cysteine residues in the disulfide bonds formed by cysteines 7-31, 10-60, 28-82, 59-87, and 32-84 (Lapthorn, A., Harris, D. Littlejohn, A., Lustbader, J. Canfield, R., Machin, K., Morgan, F., and Isaacs, N. (1994) Nature 369, 455-461) were converted to alanine, and these mutants were transfected alone or together with the wild-type hCG beta gene into Chinese hamster ovary cells. The alpha Cys-10, 28, 60, 82, and 84 mutants were not secreted and in most cases were degraded at a faster rate than the native subunit. In addition, these mutants failed to assemble with the hCG beta subunit. Mutants with alterations at alpha Cys-7, 31, 32, 59, or 87 were secreted and combined with the beta subunit. Heterodimers containing a 7-31 double mutant bound to human lutropin-chorionic gonadotropin receptor expressed in transfected human fetal kidney cells, and stimulated cAMP comparable to wild-type hCG. Dimers containing the beta subunit with either single mutant alpha 59, alpha 87, alpha 32, or the alpha 59-87 double mutant showed much lower affinity for the receptor than wild-type hCG. These results suggest that disulfide bonds associated with alpha 7, alpha 31, alpha 59, alpha 87, and alpha 32 are not essential for the alpha subunit to fold into a form that will combine with the hCG beta subunit and to produce a biologically active dimer. This contrasts with observations of the hCG beta subunit where all the disulfide bonds are required for efficient combination and folding (Suganuma, N., Matzuk, M., and Boime, I. (1989) J. Biol. Chem. 264, 19302-19307). In addition, the lack of secretion of some mutants reflects previous observations that proteins which do not fold correctly are rapidly degraded. Thus, alpha subunit mutants which fold properly are secreted and can form heterodimers.

Human luteinizing hormone and chorionic gonadotropin are targeted to a regulated secretory pathway in GH3 cells.

LH is a dimeric glycoprotein hormone that is stored in the anterior pituitary and is released in response to GnRH, while the placental hormone, human CG (hCG), sharing the same alpha-subunit and a related beta-subunit, is secreted constitutively. In search of a determinant that allows sorting of LH into a regulated secretory pathway, the genes encoding the common alpha- and LH/CG beta-subunits were expressed in the GH3 rat pituitary tumor cell line, which contains a regulated secretory pathway. Steady state labeling and subsequent chase experiments showed that not only LH but also hCG can be sorted to a regulated secretory pathway; after an initial period of constitutive secretion, the mature forms of both hormones containing processed oligosaccharides were stored intracellularly, and their release was stimulated by either forskolin or KCl depolarization. In Chinese hamster ovary cells, which lack a regulated pathway and are devoid of storage granules, only hormones containing unprocessed N-linked oligosaccharides were found. In GH3 cells the LH beta-subunit was partially retained in an endoglycosidase H-sensitive form, presumably in the endoplasmic reticulum; the enzyme-resistant fraction was secreted through a regulated secretory pathway. A large fraction of the hCG beta-subunit was released constitutively, although some mature hCG beta-subunit accumulated in secretory granules and was released by forskolin. The common alpha-subunit was secreted constitutively with little intracellular accumulation of the mature forms. We conclude that the LH beta-subunit contains sufficient information to direct LH to a regulated pathway, and alpha:LH beta assembly is not a prerequisite for this targeting. The sorting of hCG to a regulated pathway in GH3 cells presumably reflects a structural similarity between LH and hCG. In addition, we have shown that GH3 cells can recognize the N-linked oligosaccharides on the gonadotropin subunits as substrates for sulfation.