Transcription factor AP1 is involved in basal and okadaic acid-stimulated activity of the human PRL promoter.

The tumor promoter, okadaic acid (OA), an inhibitor of protein phosphatases, stimulates the activity of the human PRL (hPRL) proximal promoter. We analyzed in detail the effects of OA on transcription factor binding to elements P1 and P2 of this promoter, sequences known to contain at least one Pit-1 binding site each. OA treatment induces binding of an AP1-related transcription factor to the P1 site. This effect is specific, as protein binding to the P2 site is not altered by the treatment. Specific antibodies were used to confirm that the OA-induced complex is related to AP1 and to show that it contains JunD and c-fos, but not Pit-1. The increase in AP1 binding to P1 and to a canonical AP1 site correlates to an increase in cellular JunD and c-fos content. Transient transfection experiments showed that both AP1 and Pit-1 are involved in the regulation of basal and OA-stimulated promoter activity. Our results demonstrate that a member of the AP1 family, containing JunD and c-fos, can bind to the proximal element P1 within the hPRL promoter. In addition, they show that AP1 is involved in both basal and OA-stimulated expression of the hPRL gene.

Thyroid hormone inhibits the human prolactin gene promoter by interfering with activating protein-1 and estrogen stimulations.

Transcription of the human PRL (hPRL) gene in the pituitary is subject to tissue-specific and multihormonal regulation involving two main regulatory regions, a proximal promoter and a distal enhancer. In this report we show that thyroid hormone inhibits the expression of the hPRL gene in rat pituitary cells. Transient expression experiments show that thyroid hormone regulation involves a strong inhibitory element, located in the proximal (-164/-35) promoter, which is modulated by a more distal stimulatory response control region. Gel retardation experiments reveal that the thyroid hormone receptor does not bind to the proximal negative element. We show the existence of an activating protein-1 (AP-1) response element located at positions -61 to -54 of the proximal promoter, conferring AP-1 stimulation to the hPRL promoter. This AP-1 induction is abolished when hormone-bound thyroid hormone receptor is present, indicating that there is an interference between the thyroid hormone receptor and AP-1 regulatory pathways. Furthermore, using the complete hPRL upstream region, we show that estrogen induction is abolished by simultaneous thyroid hormone treatment.

Protein kinase C regulation of prolactin gene expression in lactotroph cells: involvement in dopamine inhibition.

The role of protein kinase C (PKC) on dopamine inhibition of PRL messenger RNA (mRNA) levels was studied in anterior pituitary cells kept in primary culture. PKC was desensitized by long-term exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA). Effectiveness of PKC desensitization was confirmed by the fact that after TPA pretreatment, short-term (1-h) exposure to TPA was no longer able to trigger PRL release. In contrast, the capacity of nonreceptor-mediated secretagogues as depolarization with 48 mM K+ to release the hormone was preserved. Pretreatment with TPA did not affect basal PRL mRNA levels. In contrast, it significantly reduced the dose-dependent inhibition of PRL mRNA induced by 1 nM bromocriptine after a 4-day incubation period. Since dopamine inhibition of PRL release is mediated by several second messager pathways, including cAMP, inositol phosphates, and Ca2+, we investigated whether PKC depletion was able to interact with direct stimulation of these pathways. Pretreatment with PKC suppressed stimulation of PRL mRNA levels induced by either Forskolin (FK) or 8Br-cAMP. In parallel, it reduced basal as well as FK stimulated intracellular cAMP levels. In addition, chronic exposure to TPA completely suppressed PRL mRNA inhibition induced by nifedipine, a dihydropyridine antagonist which blocks voltage-dependent Ca2+ channels. TPA desensitization also affected the action of bromocriptine, FK or nifedipine on PRL release measured under the same conditions. The data indicate that endogenous PKC can interfere with the regulation of PRL gene expression induced by both cAMP and Ca2+ pathways, two second messengers associated with the action of dopamine in lactotroph cells.

Alteration of G alpha subunits mRNA levels in bromocriptine resistant prolactinomas.

Patients with prolactinoma are commonly treated with the D2 dopamine agonist bromocriptine, which in most cases, normalizes prolactin (PRL) levels. However, resistance to bromocriptine has been observed in 5 to 18% of tested prolactinomas and is associated to a decrease in both D2 receptor density and mRNA levels. In this study, we used quantitative RT-PCR to investigate whether expression of G alpha proteins could be also modified in bromocriptine resistant prolactinomas. No difference in G alpha o mRNA levels or in the relative expression of G alpha s between bromocriptine sensitive and bromocriptine resistant prolactinomas was observed. In contrast, the relative expression of G alpha i2 was found to be decreased in bromocriptine resistant prolactinomas when compared to that of bromocriptine sensitive prolactinomas. Interestingly, the relative G alpha i2 expression was correlated to both bromocriptine inhibition of in vitro PRL secretion and D2 receptor mRNA levels. Bromocriptine resistance could thus result from a decrease in D2 dopamine receptors associated with a decrease in G alpha i2 expression.

[Etiopathogenesis of pituitary tumors]. / Etiopathogénie des tumeurs hypophysaires.

In this present work, the authors discuss some recent advances in the pathogenesis of pituitary tumours. The model of transgenic mice suggest that chronic hormonal stimulation and some growth factors could sustain pituitary tumour development. However, these data are not suitable for human pituitary adenomas. The evidence that most pituitary adenomas are monoclonal in origin has prompted a search for somatic mutations. The mutated Gs alpha are found in only 30-40% of somatotroph adenomas and the ras mutations seem to be associated with the malignant transformation. In some prolactinomas resistant to the bromocriptine treatment, quantitative and qualitative alterations of the dopamine receptor D2, have been described. Mutations of protein kinase C have been identified in some invasive pituitary tumours. Molecular abnormalities have been reported in some cases (allele loss at the 11q13 locus, retinoblastoma gene mutation, aberrant expression of hst gene, Pit-1 overexpression) but none by itself can explain the tumour formation. The pituitary tumorigenesis is certainly a multistep process with the intervention of multiple promoting factors.

[Membrane receptors and coupling proteins in adenohypophyseal cells]. / Récepteurs membranaires et protéines de couplage dans les cellules adénohypophysaires.

Dopamine receptors of D2 type present on lactotroph cells are coupled to a large series of transduction mechanisms. Beside their negative coupling with adenylate cyclase, they are also coupled with potassium and calcium channels, leading to a decreased intracellular calcium concentration. In addition, D2 dopamine receptors also modulate phospholipase activities. Dopamine inhibits inositol phosphate production, through two distinct mechanisms. One of them could represent a direct negative coupling with phospholipase C. All these transduction mechanisms of the D2 dopamine receptors implicate G proteins sensitive to pertussis toxin. In contrast, these receptors are negatively coupled to phospholipase A2 through G proteins insensitive to this toxin. Both isoforms of the D2 dopamine receptor, generated by alternate splicing of a single gene are present in lactotroph cells. After transfection in CH4C1 cells the two isoforms are coupled with adenylate cyclase while only the shortest isoform appears negatively coupled to phospholipase C. Functional D2 dopamine receptors are present in human prolactinomas. Resistance to bromocriptine therapy is associated with a decrease density of these receptors in the tumor. In addition, the ratio of the two receptor isoforms (measured by PCR) is different in responsive and resistant tumors. Furthermore, the activity of Gi/Go proteins coupled to adenylate cyclase appears also affected in resistant tumors. Resistance to bromocriptine therapy appears thus to involve multiple changes at the different levels of the multiple mechanisms of action of dopamine on lactotroph cells.

D2 dopaminergic receptors: normal and abnormal transduction mechanisms.

Dopamine receptors of D2 type present on lactotroph cells are coupled to a large series of transduction mechanisms. Beside their negative coupling with adenylate cyclase, they are also coupled with potassium and calcium channels, leading to a decreased intracellular calcium concentration. In addition, D2 dopamine receptors also modulate phospholipase activities. Dopamine inhibits inositol phosphate production, through two distinct mechanisms. One of them could represent a direct negative coupling with phospholipase C. All these transduction mechanisms of the D2 dopamine receptors implicate G proteins sensitive to pertussis toxin. In contrast, these receptors are negatively coupled to phospholipase A2 through G proteins insensitive to this toxin. Both isoforms of the D2 dopamine receptor, generated by alternate splicing of a single gene, are present in lactotroph cells. After transfection in CH4C1 cells the two isoforms are coupled with adenylate cyclase while only the shortest isoform appears negatively coupled to phospholipase C. Functional D2 dopamine receptors are present in human prolactinomas. Resistance to bromocriptine therapy is associated with a decreased density of these receptors in the tumor. In addition, the ratio of the two receptor isoforms (measured by PCR) is different in responsive and resistant tumors. Furthermore, the activity of Gi/Go proteins coupled to adenylate cyclase appears also affected in resistant tumors. Resistance to bromocriptine therapy appears thus to involve multiple changes at the different levels of the multiple mechanisms of action of dopamine on lactotroph 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.

Decreased expression of the two D2 dopamine receptor isoforms in bromocriptine-resistant prolactinomas.

Bromocriptine or other dopamine agonists are usually effective for the treatment of prolactin-secreting adenomas. Five to 18% of prolactinomas, however, do not respond to such therapy. We have shown previously that such resistance to bromocriptine correlates with reduced binding to the D2 receptor subtype of dopamine, the major PRL inhibiting factor. In the present work, we demonstrated that reduced binding actually corresponds to decreased expression of the gene coding for the D2 receptor in the pituitary from bromocriptine-resistant patients, as shown by 4-fold lower levels of the corresponding mRNAs compared to those coding for actin. The existence of two D2 receptor isoforms, D2S and D2L generated by alternative splicing, has been described in several tissues, including the pituitary. Both are negatively coupled to adenylyl cyclase and inhibit prolactin secretion, but, in addition, the shortest one (D2S) is more efficiently coupled to phospholipase C. Consequently, we also investigated whether expression of a particular D2 receptor isoform was preferentially affected in resistant adenomas. The proportion of messengers corresponding to the short receptor isoform (D2S) was lower in resistant compared to responsive adenomas: D2S/D2L = 0.74 +/- 0.08 and 1.00 +/- 0.07, respectively. In parallel, much lower levels of D2 receptor mRNAs were found in growth hormone-secreting adenomas, with a D2S/D2L ratio comparable to those of both normal human pituitary and bromocriptine-sensitive prolactinomas (1.05 +/- 0.11). Thus, resistance to bromocriptine therapy seems to involve defects in D2 dopamine receptor expression and possibly in posttranscriptional splicing.

Neonatal induction of tolerance to T(h)2-mediated autoimmunity in rats.

Brown-Norway (BN) rats are highly susceptible to drug-induced immune dysregulations and when injected with mercuric chloride (HgCl(2)) or sodium aurothiopropanolsulfonate (ATPS), they develop a syndrome characterized by a polyclonal B cell activation depending upon CD4(+) T(h)2 cells that recognize self-MHC class II molecules. Since peripheral tolerance of T(h)2 cells might be crucial in the prevention of immunological manifestations such as allergy, establishing conditions for inducing tolerance to HgCl(2)- or ATPS-mediated immune manifestations appeared to be of large interest. We report here that BN rats neonatally injected with HgCl(2): (i) do not develop the mercury disease, (ii) remain resistant to HgCl(2)-induced autoimmunity at 8 weeks of age and later, provided they are regularly exposed to HgCl(2), (iii) are still susceptible to ATPS-induced immune manifestations, and (iv) exhibit spleen cells that adoptively transfer tolerance to HgCl(2)-induced autoimmunity in naive, slightly irradiated, syngeneic recipients. These findings demonstrate that dominant specific tolerance can be neonatally induced using a chemical otherwise responsible for T(h)2-mediated autoimmunity.

Normal IgG protects against acute graft-versus-host disease by targeting CD4(+)CD134(+) donor alloreactive T cells.

Intravenous immunoglobulin (IVIg) was shown to decrease the severity of acute graft-versus-host disease (aGVHD) in recipients of allogeneic bone marrow transplants. To investigate the mechanisms involved in the protective effect of IVIg, we have used the parent-into-F1 model in which parental lymphocytes are transferred into semi-syngeneic non-irradiated F1 rats. Here we report that IVIg, as well as F(ab')(2) fragments of IVIg, protected (Lewis x Brown-Norway) F1 rats against aGVHD induced by a single injection of Lewis lymphocytes. IVIg was given as five consecutive daily injections, starting on the day preceding that of the transfer of Lewis cells. Protection was associated with a decreased ability of lymphocytes to spontaneously proliferate and to produce NO and IFN-gamma, in the absence of an increased production of IL-10. We further demonstrate that protection was associated with a decrease in CD4(+) T cells bearing the activation marker CD134 in vivo, and with an enhanced apoptosis of activated CD4(+) T cells by IVIg, in vitro. Our observations suggest that the prevention of aGVHD by IVIg in this model is mediated by the induction of apoptosis of activated alloreactive CD4(+)CD134(+) donor T cells. The results further emphasize the role of normal immunoglobulin in modulating alloantigen immune responsiveness.