We cloned the 5-flanking region of the human growth hormone-releasing hormone receptor (GHRH-R) gene and determined the nucleotide sequence of 2.7 kilobases upstream from the translation start site. RNase protection analysis showed the major transcription start site is 122 base pairs upstream from the translation start site. The 5-end of the longest product of 5-rapid amplification of cDNA ends was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-1-binding site-like element. Transient transfection studies using a luciferase reporter gene demonstrated that 5-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in nonpituitary cells, BeWo and HeLa cells. However, co-transfection of Pit-1 expression vector increased luciferase activity in BeWo cells. Deletion study showed that the regions from ؊310 to ؊130 and from ؊130 to ؊120 were important for the GHRH-R gene expression in GH3 cells, although the latter contributed less to the gene expression. In BeWo cells cotransfected with Pit-1 expression vector, the region from ؊310 to ؊130 was essential for the Pit-1-dependent expression of GHRH-R gene. The region from ؊310 to ؊120 has two putative Pit-1-binding sites, P1 and P2, located from ؊129 to ؊123 and from ؊171 to ؊160, respectively. Both mobility shift assay and DNase-I footprint analysis showed that P2 had much higher Pit-1 binding affinity than P1. Mutation of P2 decreased GHRH-R gene expression in GH3 cells. These findings were consistent with the results that the region from ؊310 to ؊130 is an important element for Pit-1-dependent expression of GHRH-R gene. Growth hormone-releasing hormone (GHRH)1 plays a major role in stimulating both synthesis and release of growth hormone (GH) in the anterior pituitary through its specific receptor. So far, the GHRH receptor (GHRH-R) has been cloned in human (1, 2), mouse (3), rat (1), and pig (4). The GHRH-R is a member of G protein-coupled receptor family and transduces GHRH-dependent increase in intracellular cAMP via G s activation for stimulating somatotroph proliferation and GH gene expression (1-4). Lin et al. (5) demonstrated that one amino acid substitution of the GHRH-R in the little mouse, showing genetically transmitted dwarfism, caused GH deficiency and somatotroph hypoplasia. In addition, an amber-type mutation (Glu72Stop) of the GHRH-R in humans was demonstrated to cause profound GH deficiency (6). These genetic disorders suggest the physiological significance of GHRH-R in hypothalamopituitary GH axis.The relationship between the dynamics of GHRH-R expression and GH secretion remains to be clarified. Not only the functional defects of GHRH-R but also the amount of GHRH-R should affect GH synthesis and secretion in the pituitary. Hypothalamic hormone, neurotransmitters, various hormonal states, and nutrition all could modulate the activity of the GHRH-GH axis. For instance, glucocorticoids potentiate GHRH action and enhance GH secretion in rats (7-9), where...

Pit-1 stimulates the expression of growth hormone, prolactin, and thyrotropin ␤ subunit genes. Consequently, abnormality of the Pit-1 gene results in combined pituitary hormone deficiency (CPHD). In this study, we analyzed the function of Pit-1 with a mutation (proline to leucine at codon 24) in the transactivation domain, P24L, which has a normal POU domain important for binding to DNA, because this mutation had been reported in a patient with CPHD. We found that codon 24 proline in the transactivation domain as well as the POU domain of Pit-1 was crucial to recruit coactivator CREB-binding protein (CBP) in the cultured cells. P24L completely lost the responsiveness to cAMP to stimulate the expression of the Pit-1-targeted genes. Furthermore, CBP and Pit-1, but not P24L, markedly enhanced the expression of the Pit-1-targeted gene to cAMP, and adenovirus E1a that binds to CBP and abrogates its function blocked the induction by cAMP of Pit-1-stimulated gene transcription in the pituitary-derived GH3 cells. These results suggest that CBP and proline at codon 24 in the transactivation domain of Pit-1 are important for the cAMP-induced activation of Pit-1-targeted genes. However, P24L maintained basal transcriptional activity, suggesting that CBP is unlikely to be an essential coactivator for Pit-1.

Abstract.Multifocal fibrosclerosis denotes a combination of similar fibrous disorders occurring at different anatomical sites. We encountered a 53-year-old male patient with orbital pseudotumor, chronic paranasal sinusitis, fibrous nodules of the lungs, intracranial pachymeningitis, and panhypopituitarism with central diabetes insipidus (DI) as a possible manifestation of multifocal fibrosclerosis. It has been reported that intracranial pachymeningitis or orbital pseudotumor associated with multifocal fibrosclerosis could invade the sella turcica causing a variety of anterior and/or posterior pituitary dysfunctions.In our case, intracranial pachymeningitis apparently involved the pituitary stalk and gland. Isolated gonadotropin deficiency, in addition to central DI, preceded panhypopituitarism. Although panhypopituitarism with central DI due to multifocal fibrosclerosis is quite rare and only one case has ever been reported, this systemic fibrotic disorder can be a possible cause of panhypopituitarism with central DI.

Wedescribe a 52-year-old male manifesting severe insulin resistance associated with hypereosinophilic syndrome (HES). Diabetes mellitus was initially well-controlled by an oral hypoglycemic agent, and thereafter by humaninsulin. Due to the progression ofhypereosinophilia, hepatosplenomegaly and peripheral lymphoadenopathy, severe insulin resistance associated with diabetic ketoacidosis occurred repeatedly, despite intravenous administration of over 1,000 U per day of human insulin. A high plasma insulin-binding capacity as determined by Scatchard analysis was consistent with insulin antibody-mediated resistance. The diagnosis of HESwas made due to the persistent elevation ofeosinophil count and associated liver and cardiac damage. Glucocorticoid therapy successfully achieved both reducing clinical symptoms and improving glycemic control. (Internal Medicine 33: 632-636, 1994)

Objective: Genetic abnormalities of the pituitary specific transcription factor, Pit-1, have been reported in several patients with GH, prolactin (PRL) and TSH deficiencies. The most common is a mutation altering an arginine to a tryptophan in codon 271 (R271W) in one allele of the Pit-1 gene. According to the previous in vitro expression study, R271W acted as a dominant negative inhibitor of the wild type to activate the GH promoter. However, healthy carriers with this mutation, who should be affected by the dominant negative effect of R271W, have also been reported. The aim of this study was to clarify in more detail the function of this mutant form of Pit-1. Methods: Transcriptional activity of R271W for the expression of Pit-1-associated genes was investigated in COS7 cells with the aid of transient transfection assays. The 1.8 kb rat GH, 0.6 kb rat PRL or 1.9 kb rat PRL 5 0 -flanking regions were inserted upstream of the luciferase reporter gene and were used for functional analysis of R271W. Another reporter gene containing seven Pit-1 responsive elements was also used. The same experiments were also performed using JEG3 and CHO cells. Results: We could not confirm the dominant negative effect of R271W on wild type Pit-1. Furthermore, our expression study revealed that R271W could activate the promoters of GH and PRL genes to levels similar to the wild type. Conclusion: Taken together with the evidence that phenotypically normal cases have been reported with this mutation, our results deny the relationship between R271W and combined pituitary hormone deficiency.