Combined pituitary hormone deficiency due to the F135C human Pit-1 (pituitary-specific factor 1) gene mutation: functional and structural correlates.

The pituitary-specific transcription factor Pit-1 (pituitary-specific factor 1) is known to play a key role in the differentiation of PRL-, GH-, and TSH-secreting cells, and in the regulation of expression of the corresponding genes. In recent years, 12 distinct mutations of the Pit-1 gene have been shown to be responsible for a phenotype of multiple congenital pituitary hormone deficiency involving PRL, GH, and TSH. We had previously identified, in four siblings with GH, PRL, and TSH deficiencies, a mutation (F135C) resulting in a single amino acid change within the POU-specific binding domain of the Pit-1 molecule. In the present report, we have explored the functional effect of the F135C mutation. In vitro activity tests performed by transfection in human HeLa cells showed decreased transactivation capacity on the PRL, GH, and Pit-1 genes. The DNA binding experiments performed by gel shift showed that the F135C mutation generated a protein capable of binding to DNA response elements. To analyze how the F135C mutation might affect functionality of the transcription factor despite a normal DNA binding, we used a structure modelization approach and also analyzed two other Pit-1 mutant proteins (F135A and F135Y). The loss of functionality in these two mutants was similar to that of F135C. This finding was in keeping with our molecular modeling studies. According to structural data derived from the crystallographic analysis of the DNA/Pit-1 POU domain complex, the conformation of the first helix of the F135C-mutated POU-specific domain could be perturbed to such an extent that any interaction with other transcription cofactors might be definitively prevented.

Impact of gsp oncogene on the expression of genes coding for Gsalpha, Pit-1, Gi2alpha, and somatostatin receptor 2 in human somatotroph adenomas: involvement in octreotide sensitivity.

The impact of the gsp oncogene on the expression of genes engaged in the somatotroph cell phenotype remains poorly understood in human somatotroph adenomas. As the gsp oncogene is associated with an increased octreotide (somatostatin agonist) sensitivity, a group of 8 somatotroph adenomas bearing the gsp mutation (gsp+) and another group of 16 adenomas without the mutation (gsp-) were analyzed, all of them presenting variable octreotide sensitivities. The expressions of genes encoding for G(s)alpha, Pit-1, G(i2)alpha, and SSTR2, involved in the regulation of secretory activity in somatotroph cells, were assessed by Northern blot. A decreased expression of the G(s)alpha gene was found in gsp + tumors, suggesting the existence of a negative feedback of the oncogenic protein upon its own messenger ribonucleic acid (mRNA). In contrast, G(i2)alpha, Pit-1, and GH messengers were not significantly different in the groups. A positive correlation between the in vitro and in vivo GH octreotide-induced secretory inhibition and the expression of SSTR2 mRNA was found. However, the expression of the gene for SSTR2 appeared not to be different between gsp + and gsp-, even when the octreotide sensitivity was significantly higher in the adenomas carrying the mutation. Interestingly, the SSTR2 gene expression was significantly correlated to those of G(i2)alpha and Pit-1. In the same way, the G(s)alpha mRNA expression was positively correlated with those of Gi2alpha and Pit-1. Such correlations strongly suggest a concerted dysregulation of the expression of these genes in both categories of adenomas. The loss of the octreotide sensitivity represents one aspect of the dysregulation process that partially results from the decreased SSTR2 expression. However, the improvement of the sensitivity associated with the presence of the gsp oncogene seems to proceed in a way different from SSTR2 expression.

A new mutation of the gene encoding the transcription factor Pit-1 is responsible for combined pituitary hormone deficiency.

The pituitary-specific transcription factor Pit-1/GHF1 regulates the expression of PRL, GH, and TSH beta genes through binding to specific regions of the promoters of these genes. Mutations of the Pit-1 gene have been shown to be responsible for a syndrome of combined pituitary hormone deficiency (CPHD), including complete GH and PRL deficiencies and central hypothyroidism. We studied four siblings presenting with CPHD born to healthy consanguinous parents. All four affected children had complete GH deficiency diagnosed in early childhood. They later developed hypothyroidism and were found to have undetectable PRL levels. The pituitary gland was hypoplastic at magnetic resonance examination in one of the patients. Amplification of genomic DNA and subsequent sequencing of the six exons of the Pit-1 gene allowed identification in the four patients with CPHD of an as yet undescribed mutation in exon 3. A substitution of T go G induced a change from a Phe to a Cys residue at position 135 within the hydrophobic core of the POU-specific DNA-binding domain of the Pit-1 protein. All affected children were homozygous for the mutation, whereas the mother was heterozygous, suggesting a recessive mode of inheritance. Molecular studies in other affected families will allow instructive genotype-phenotype correlations concerning the Pit-1 gene.

Pit-1 gene expression in human lactotroph and somatotroph pituitary adenomas is correlated to D2 receptor gene expression.

The expression of the pituitary-specific transcription factor Pit-1 gene was analyzed in a series of 30 human lactotroph and somatotroph pituitary tumors. Northern blot analysis failed to reveal any quantitative differences in Pit-1 gene expression between somatotroph and lactotroph tumors, and reverse transcription-PCR analysis showed similar patterns of Pit-1 isoforms expression in both populations of tumors. The expression of the D2 receptor gene was subsequently analyzed in the same adenomas. In the prolactinomas, which presented with a variable sensitivity to dopamine agonist treatment, the intensity of the D2 receptor transcripts (2.8 kilobases) was variable and was related to the sensitivity to the dopamine agonist treatment. Notably, the individual D2 receptor messenger ribonucleic acid (mRNA) levels were highly correlated to the Pit-1 mRNA levels measured in the same tumors (r = 0.90; P < 0.0001). In the GH-secreting tumors, a significant expression of the D2 receptor gene was evidenced by Northern blot in all mixed somato-lactotroph adenomas and in some of the pure somatotroph adenomas; again, a positive correlation was found between D2 mRNA and Pit-1 mRNA levels (r = 0.68; P < 0.01). These results suggest the existence of mechanisms responsible for a coordinate control of Pit-1 and D2 receptor genes that remain to be determined.

Expression of the members of the Ptx family of transcription factors in human pituitary adenomas.

A number of putative transcription factors described in the pituitary have been implicated as key elements in the processes that direct pituitary development. Three recently described proteins, Ptx1, Ptx2, and Ptx3, define a new family of transcription factors, the Ptx subfamily, within the paired-like class of homeodomain factors. In mice, Ptx1 and Ptx2 gene expression has been detected in the area of the pituitary primordium and is maintained throughout development in Rathke pouch and adult pituitary. In the present study, the expression of the Ptx1, Ptx2, and Ptx3 genes was characterized in the normal human pituitary and in the different types of human pituitary adenomas. Although no Ptx3 gene expression could be detected in these tissues, Ptx1 presented with a quite ubiquitous pattern of distribution, being expressed at quite constant levels in normal tissues and in all 60 pituitary tumors analyzed. The pattern of expression of the Ptx2 gene among the different subsets of pituitary adenomas was even more varied. No Ptx2 expression could be detected in corticotroph tumors. In contrast, high levels of Ptx2 messenger ribonucleic acid were measured in the gonadotroph tumors, although no specific correlation to other markers of the gonadotroph lineage differentiation, such as alphaGsu, LHbeta, or FSHbeta, could be evidenced. Finally, Ptx2 was also expressed in pure lactotroph adenomas and not in somatotroph adenomas. Ptx2 is, therefore, the first paired homeodomain pituitary transcription factor differentially expressed in these two lineages, which derive from a common precursor. These results support a role for Ptx2 in the terminal differentiation of somatotroph and lactotroph cell phenotypes.

PROP1 gene screening in patients with multiple pituitary hormone deficiency reveals two sites of hypermutability and a high incidence of corticotroph deficiency.

Alterations of the gene encoding the pituitary transcription factor PROP1 were associated with congenital forms of multiple pituitary hormone deficiencies in several families. Among 23 patients with multiple pituitary hormone deficiencies screened for a PROP1 gene abnormality, nine belonging to eight unrelated families had homozygous PROP1 gene defects. All mutations were located in exon 2 and affected only two different sites: a homozygous AG deletion at codons 99/100/101 (n = 5); homozygous point mutations affecting codon 73: R73C (n = 2) or R73H (n = 1), and a R73C/R99X double-heterozygous mutation (n = 1). R73H and R99X were never described. All patients were born to unaffected parents, and consanguinity was documented in two patients. They had complete GH, LH-FSH, and TSH deficiencies and normal basal levels of PRL. Delayed ACTH deficiency was diagnosed in four of nine patients. At magnetic resonance imaging the anterior pituitary was hypoplastic in seven patients and hyperplastic in two. This study found two novel mutations (R73H and R99X) and underlines the high incidence of PROP1 gene alterations in patients with multiple pituitary hormone deficiencies. A corticotroph deficiency was frequently observed in association with GH, TSH, and gonadotropin deficiencies and should be carefully sought during follow-up.

Expression of functional growth hormone secretagogue receptors in human pituitary adenomas: polymerase chain reaction, triple in-situ hybridization and cell culture studies.

We examined the expression of functional growth hormone secretagogue receptors (GHS-R) in a series of 30 human pituitary adenomas-six secreting GH, three GH-PRL, six prolactin (PRL), five adrenocorticotrophic hormone (ACTH), one thyroid stimulating hormone (TSH), four gonadotroph and five non-secreting adenomas. By reverse transcriptase polymerase chain reaction (RT-PCR), the coexpression of the two GHS-R isoforms (Ia and Ib) was found in all the GH-, GH-PRL- and PRL-secreting adenomas, and only in two out of three corticotroph, two out of four gonadotroph and one out of five non-secreting tumours. They were absent in the TSH-secreting adenoma. The PCR products of GHS-R Ia and Ib were identical in size to those from two normal pituitaries. PCR cloning and sequencing of isoforms performed in two somatotroph adenomas revealed only two single, silent base mutations. Triple in-situ hybridization showed colocalization of GHS-R mRNA with messengers of GH and PRL, conjointly or separately, in individual cells of somatotroph, mammosomatotroph, and lactotroph adenomas. The presence of GHS-R mRNA in cells expressing PRL mRNA is emphasized. In cultured cells from six somatotroph and two mammosomatotroph adenomas, the powerful GHS MK-0677 stimulated GH release in a dose-dependent manner, with maximal effect at 6 h. Contrarily, when GHRH was applied, only three somatotrophs and two mamosomatotrophs were stimulated. In the two mammosomatotrophs, the PRL response to MK-0677 and to GHRH was similar to the GH response. An homologous desensitization of the GHS-R and the GHRH receptor was observed 24 h after a first stimulation by a single dose of the corresponding agonist. Heterologous desensitization was not observed. Interestingly, MK-0677 also stimulated, in a dose-dependent way, the hormone release of cells from all tested lactotroph and corticotroph adenomas. The existence of a functional expression of GHS-R in somatotroph, mammosomatotroph, lactotroph and corticotroph adenomas rises the question of the role played by GHS-R in pituitary adenomas, particularly those not engaged in GH secretion.

[Homeoproteins and pituitary adenoma]. / Homéoprotéines et adénomes hypophysaires.

Several transactivating factors specifically involved in the differentiation and proliferation of anterior pituitary cell types have been recently identified. Among them Pit-1 a member of the POU-domain transcription factors family is specific of anterior pituitary cells, and was initially identified and cloned as a transactivator of the GH and PRL genes and as a regulator of the TSH beta gene. Pit-1 play a key role during embryogenesis in the differentiation and proliferation of somatotrophs, lactotrophs and thyreotrophs. The importance of Pit-1 as a regulator in the anterior pituitary development has been further demonstrated by naturally occurring mutations or delections in dwarf mouse strains. In the Snell and Jackson dwarf mice, the levels of Pit-1 gene expression are low or undetectable, GH, PRL and TSH beta gene expression are absent and lactotrophs, somatotrophs, and threotrophs fail to proliferate. Furthermore Pit-1 carries out similar functions in humans. This is supported by the fact that children with mutations of the Pit-1 gene present with a congenital combined GH, PRL and TSH deficiency analogous to the phenotype of the Snell and Jackson dwarf mice. In children who were born to healthy consanguinous parents and present such combined deficiencies we recently reported a Pit-1 mutation causing a transition from a Phe to a Cys in a region of the protein known to be involved in DNA binding. Pit-1 transcripts identical in size and sequence to those observed in normal pituitary were described in human GH, PRL and TSH secreting pituitary adenomas. The Pit-1 beta isoform, raised through alternative splicing of exon 2 of the Pit-1 gene, is a more potent inducer of GH transcription than the major Pit-1 form. However no difference in the level of expression of the different Pit-1 isoforms was observed between tumors identified as pure GH or PRL producing tumors. The results support the existence of other transcription factors interacting with Pit-1 to coordinately regulate the activity of the GH and PRL promotors in a cell specific manner. In contrast, variable Pit-1 expression was observed in prolactinomas, according to their sensitivity to bromocriptine treatment. A highly significant correlation was indeed evidenced between the D2 receptors mRNA and the Pit-1 mRNA levels. These results raise the possibility that Pit-1 may either directly or indirectly affect the transcription of the D2 dopaminergic receptor gene. In fact, receptors for other hypothalamic neurohormones such a GHRH and somatostatin are known to be potential Pit-1 target genes. Such mechanisms could be implicated in the differentiation and proliferation of lactotrophs and somatotrophs.

[Congenital multiple anterior pituitary hormone deficiencies. An approach of pituitary ontogenesis]. / Les déficits anté-hypophysaires associés congénitaux. Une approache de l'ontogenèse hypophysaire.

A number of transcription factors evidenced in the pituitary may play a part in the development of this gland, namely Pit-1, Prop-1, P-Lim, Ptx1, Rpx, Dax-1, SF-1. Several of these factors are involved in animal models of hypopituitarism, while up to now only Pit-1 gene alterations have been shown to be responsible for hypopituitarism in man. These factors are briefly presented, and current data on genetically determined uni- or multi-hormonal pituitary deficiencies are reviewed. In particular, data on combined somatotroph, lactotroph and thyrotroph deficiencies due to Pit-1 gene alterations are detailed. Analysis of phenotype-genotype relationships in this syndrome and in other pathological models of multiple pituitary hormone deficiencies will provide useful information on the complex sequence of events that contribute to the development of the pituitary gland and to differentiation and regulation of the different pituitary cell lines.

[Transcription factors of the anterior pituitary and combined hypopituitarism]. / Facteurs de transcription anté-hypophysaires et hypopituitarismes multiples.

In the recent years, spontaneous and experimental models of hypopituitarism have underlined the involvement of a number of homeodomain transcription factors in different forms of congenital anterior pituitary hormone deficiencies. Indeed, abnormalities of the transcription factor Pit-1 are responsible for combined deficiencies affecting thyrotroph, somatotroph and lactotroph cell lineages both in dwarf mouse strains (Snell, Jackson) and in human patients. More recently, alterations of the Prop-1 gene have been shown to induce a similar phenotype in the Ames mice, and alterations of the human homolog gene have been evidenced in patients with anterior pituitary deficiencies involving the same three lineages together with the gonadotrophs. Gene knock-out experiments have demonstrated the importance of other transcription factors such as Lhx3 and Lhx4 in the development of the normal pituitary gland. These findings illustrate the potential involvement of anomalies of these and many other factors in the various forms of multiple hypopituitarism.

[Pituitary development and pathology of transcription factors]. / Développement hypophysaire et pathologie des facteurs de transcription.

Over the last 10 years, important data on pituitary development have been reported using spontaneous or experimental models of gene inactivation. The development pathways of the anterior pituitary lobe include organogenesis resulting in Rathke pouch formation and cell differentiation. Pituitary development is controlled by sequential series of specific signaling molecules and specific transcription factors. In humans, inactivation of Pit-1, Prop-1, Rpx and Ptx2 genes is responsible for pituitary combined deficiency. In this paper, updated data on the embryology of the pituitary gland and the clinical phenotypes of inactivation of pituitary transcription factors are reported.

Pit-1 gene expression in human pituitary adenomas.

The anterior pituitary-specific transcription factor Pit-1 (also known as GHF-1) was initially identified and cloned as a transactivator of the GH and PRL genes, and later as a regulator of the TSH beta gene. Analysis of Pit-1 expression during mouse embryogenesis revealed that initiation of its expression correlates both temporally and spatially with activation of its target genes. Immunocytochemical studies revealed a high expression of Pit-1 protein in the nuclei of only three cell types: somatotropes, lactotropes and thyrotropes. The importance of Pit-1 as a regulator of the anterior pituitary development has been further demonstrated by the absence of somatotropes, lactotropes and thyrotropes in the pituitary glands of Pit-1-defective mice and humans. Since Pit-1 is required for both cell phenotype and proliferation, one may ask if this transcription factor might be associated with development of pituitary tumors. Several investigators have reported Pit-1 gene expression in human pituitary adenomas. These studies, while not in total agreement, show that pituitary tumorigenesis does not seem to be associated with a gross alteration of Pit-1 gene expression in humans. Pit-1 transcripts, identical in size and sequence to those observed in normal pituitary, were described in human GH-, PRL- and TSH-secreting pituitary adenomas and in most cases the presence of Pit-1 transcripts correlated with the localization of Pit-1 protein. The biological relevance of Pit-1 expression reported in some nonfunctioning adenomas remains to be clarified. As expression of the PRL and GH genes is ultimately confined to distinct lactotropic and somatotropic populations despite the presence of Pit-1 protein in both cell types, there must be additional mechanisms that control the cell-specific activation of the PRL and GH promoters. The Pit-1 beta isoform, raised through alternative splicing of exon 2 of the Pit-1 gene, is a more potent inducer of GH transcription than the major Pit-1 form. This form could, at least in part, account for the cell-specific activation of the PRL and GH genes. Pit-1 beta was invariably found present in all the tumors expressing the Pit-1 major form, no significant difference in the Pit-1 beta/Pit-1 expression ratio being observed between tumors identified as pure GH- or PRL-producing tumors. This lack of selectivity together with its low level of expression is therefore not in favor of a key role for the beta-isoform in the cell type-specific expression of the GH and PRL genes in humans. The failure of somatotropes, lactotropes and thyrotropes to proliferate in Pit-1-defective mice and humans indicates that Pit-1 might be competent to activate genes required for cell proliferation or survival of the three cell types. Recent data indeed suggest that Pit-1 may directly or indirectly regulate somatotropes and lactotropes through activation of the receptors for GRF and SRIF on the one hand, and dopamine on the other hand. Such regulatory mechanisms could contribute to the differentiation of the somatomammotropic lineage in fully differentiated somatotropic and lactotropic cells.

Abnormal transduction mechanisms in pituitary adenomas.

Pituitary adenomas are differentiated tumors expressing their appropriate mature hormone. Tumoral cells sometimes present with a defective physiological inhibitory or stimulatory control, resulting in paradoxical responses or nonresponsiveness to regulatory neurohormones. These abnormalities can be explained by defects at the intracellular transduction mechanism level. Knowledge of these defective pathways has made progress in the understanding of the pathogenesis of pituitary adenomas possible. The discovery of mutations of Gs alpha named gsp oncogenes in 40% of human somatotropinomas represents one of the most important advances in this field. Other molecular alterations were identified but are rare and sporadic and the pathogenesis of pituitary adenomas remains largely unknown. Abnormal transduction mechanisms may also result in a variable sensitivity of tumors to pharmacological therapy. The dopamine agonist, bromocriptine, is able to normalize blood PRL levels and to reduce tumor size in the majority of patients with prolactinoma, but is ineffective in 8-15% of them. Under physiological conditions, PRL secretion is under the tonic inhibitory control of dopamine which binds D2 receptors negatively coupled to adenylyl cyclase. Several defects in the dopaminergic transduction pathways participate in this bromocriptine resistance. The mean D2-binding site density is decreased to 50% as compared to responsive tumors. This loss of D2 receptors can account for a lower transcription level of its gene and is accompanied by modifications in the messenger alternative splicing; the D2 short isoform receptor expression decreases preferentially. A reduction in Gi2 alpha protein expression is also observed and is correlated to that of the D2 receptor. Finally, the pituitary-specific transcription factor Pit-1 expression is affected. A highly significant correlation was seen between the D2 receptor mRNA and Pit-1 mRNA levels. These defects observed on many levels of the dopaminergic transduction cascade may be the first steps in the loss of the functional features of differentiated tumors toward more proliferative tumors.