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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 30, 2004 as doi:10.1096/fj.04-2138fje. |
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Max Planck Institute of Psychiatry, Munich, Germany
1 Correspondence: Max-Planck Institute of Psychiatry, Kraepelinstrasse 10, Munich 80804, Germany. E-mail: marcelo{at}mpipsykl.mpg.de
SPECIFIC AIMS
Sonic hedgehog (Shh) is a differentiating factor that regulates patterning and proliferation of many organs in the embryo. In mammals, Shh binds to its receptors Patched 1 (Ptc1) and Patched 2 (Ptc2), leading to activation of the Gli transcription factors. The signal is primarily mediated by Gli2 and Gli3, which activate Gli1, which in turn is responsible for the activation of the downstream target genes. Shh signaling exerts effects on cell proliferation and cell type determination in early pituitary development. The aim of this work was to investigate the expression and function of Shh in the adult pituitary gland.
PRINCIPAL FINDINGS
We show here that the Shh pathway is expressed in the anterior pituitary, where it acts in an autocrine way as well as in addition to the corticotropin-releasing hormone (CRH) pathway to increase corticotropin (ACTH) production.
1. Corticotroph cells express components of the Shh pathway
The expression of Shh, Ptc1, Ptc2, and Gli1 in the adult pituitary gland was examined by single and double immunohistochemistry. Shh was expressed in 
10% of adult anterior pituitary cells and co-localized in ACTH-immunoreactive cells. The cell types that express the other pituitary hormones did not show Shh expression. The Shh receptors Ptc1, Ptc2, and the transcription factor Gli1 were expressed in human anterior pituitary. Corticotroph cells express Ptc2 and Gli1, but not Ptc1. These results indicate that the Shh pathway is active in corticotroph cells.
2. Shh stimulates ACTH secretion
We examined the functional role of the Shh pathway in corticotroph cells using primary cultures from rat pituitary. Shh treatment for 24 h dose-dependently increased ACTH secretion. A similar effect was observed in the AtT-20 mouse corticotroph cell line. Therefore Shh stimulates ACTH production in corticotroph cells.
3. Shh and Gli1 stimulate POMC transcription
To determine whether the observed increase in ACTH production occurs at the transcriptional level, we studied the effect of Shh on the transcription of POMC, the ACTH precursor. Using a luciferase reporter, we found that Shh stimulated POMC transcription in AtT-20 cells.
Our experiments confirmed that the Shh pathway functions in corticotroph cells in the same way as observed in other tissues: Shh and Gli1 stimulate Gli-dependent transcription.
Prompted by the above results, we hypothesized that Gli1 mediates the Shh-induced increase in POMC transcription. To test this hypothesis, we transiently cotransfected AtT-20 cells with a Gli1 expression plasmid and a POMC reporter plasmid. Gli1 overexpression induced a 6-fold increase in POMC transcription. Analysis of the sequence of the POMC promoter did not detect the presence of the consensus Gli binding sites. Taken together, these results indicate that Shh stimulates Gli-dependent transcription and Gli1 indirectly stimulates POMC transcription.
4. Additive effects of Shh and CRH in ACTH secretion
CRH is the main physiological stimulus of ACTH secretion. We studied the effect of a combination of Shh and CRH on ACTH secretion by stimulating rat pituitary cells in primary culture for 24 h with Shh, CRH, or both. CRH on its own increased ACTH secretion. Interestingly, the addition of Shh produced an additive effect resulting in a significant increase in ACTH production (Fig. 1
A). We obtained similar results using the AtT-20 cell line (Fig. 1B
). Therefore, Shh and CRH act together to increase ACTH production in corticotroph cells.
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Knowing that Gli1 stimulates POMC transcription, we tested a possible cross-talk of both pathways on the POMC promoter. We transfected AtT-20 cells with the POMC reporter plasmid and stimulated them with Shh, CRH, and a combination of the two (Fig. 1C
). Shh and CRH produced an additive increase in POMC transcriptional activity. These results fit in with the additive stimulation of ACTH.
5. Cross-talk between the Shh and CRH pathways
Activation of the CRH pathway increases the intracellular levels of cAMP. We found that Shh on its own induced a slight increase in cAMP and the combination of Shh and CRH had an additive effect on intracellular cAMP levels (Fig. 1D
). This result is in line with the stimulation of ACTH by Shh.
To determine whether the two pathways cross-talk at the level of Gli, we transiently transfected AtT-20 cells with a reporter plasmid containing Gli binding sites and stimulated with Shh, CRH, or both. CRH on its own was able to stimulate Gli-dependent transcription and in combination with Shh had produced an additive effect (Fig. 1E
). Hence, there is cross-talk between CRH and Shh pathways that increases Gli-dependent transcription.
CRH acts in corticotroph cells through the CRH-R1 receptor. Therefore, the additive effects between Shh and CRH can be explained by a stimulation of the expression of CRH-R1 by Shh. To test this hypothesis, we studied the effect of Shh on CRH-R1 expression in AtT-20 cells. Shh and CRH produced a slight increase in CRH-R1 protein expression, and a combination of the two had a stronger effect (Fig. 1F
). Therefore, the stimulation of CRH-R1 by Shh could explain in part the increased response to CRH.
Our results indicate that Shh and CRH up-regulate the CRH-R1 receptor and cAMP levels. Shh and CRH stimulate Gli-dependent transcription, which leads to an increase in POMC transcription and ACTH production.
6. Gli1 inhibition reduces the CRH-induced stimulation of POMC transcription
The facts that Gli1 increases POMC transcriptional activity and is up-regulated by CRH suggested that Gli1 might be necessary to mediate the stimulation of POMC transcription produced by CRH. To test this hypothesis we used siRNA to inhibit Gli1. In AtT-20 cells cotransfected with the Gli1 reporter plasmid and Gli1 siRNA, the Gli1 siRNA produced a significant reduction in Gli-dependent transcription, to levels similar to the baseline values of the mutant Gli1 reporter plasmid (Fig. 2
A). Moreover, the Gli1 siRNA had no effect on the mutant Gli1 reporter plasmid, indicating that Gli1 siRNA specifically inhibits the transcriptional activity of Gli-1 in the transfected cells.
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To test whether Gli1 is necessary for CRH effects, we co-transfected AtT-20 cells with a POMC reporter and Gli1 siRNA, then stimulated the cells with CRH. Gli1 siRNA abolished the stimulation of POMC produced by CRH, whereas the negative control siRNA had no effect on POMC stimulation (Fig. 2B
). Hence, Gli1 is necessary for the CRH-induced increase in POMC transcription.
CONCLUSIONS AND SIGNIFICANCE
In the present study, we investigated the expression and function of Shh in the adult pituitary. We found that Shh is expressed in adult corticotroph cells. This cell type expresses the Shh receptor Ptc2 and the transcription factor Gli1. Gli1 expression requires Shh signaling, so the presence Gli1 protein in adult corticotroph cells suggests that the Shh pathway is active in this cell type. Here, Shh stimulates Gli1-dependent transcription, POMC transcription and ACTH production. These results provide the molecular basis for an autocrine mechanism involving Shh, Ptc2, and Gli1 for control of ACTH biosynthesis in adult corticotroph cells.
The physiological function of corticotroph cells in response to stress is regulated by CRH. CRH produced in the hypothalamus is a potent stimulator of POMC gene expression and ACTH production in the pituitary gland. CRH stimulates the transcription factors AP1, CREB, and Nur. These factors act directly on the POMC promoter and stimulate ACTH biosynthesis. It has been suggested that the complex regulation of POMC to coordinate the response to different stimuli may involve other factors. Our results demonstrate that the Shh pathway—in particular, Gli1—play a role in the regulation of POMC in response to CRH.
We found that Shh and CRH have additive effects on ACTH production resulting from cross-talk between the respective signaling pathways at different levels. Identifying Shh as one of the factors that may control the expression of the CRH-R1 receptor, our data contribute to elucidating the regulation of the CRH-R1 receptor. The up-regulation of CRH-R1 protein expression by Shh may explain the increase in the response of corticotroph cells to CRH. This and the additive effects with CRH could explain in part the stimulatory effects of Shh on POMC transcription and ACTH production.
Our results show further that the Shh and CRH pathways cross-talk at the intracellular level. CRH stimulates Gli-dependent transcription and Gli1 overexpression indirectly stimulates POMC transcription. We suggest that the indirect stimulation of POMC by Gli1 is a newly discovered mechanism for stimulation of ACTH biosynthesis. This is supported by the fact that the inhibition of Gli1 with siRNA blunted the response of the POMC promoter to CRH stimulation. Hence Gli1 plays a role in CRH signaling and is necessary for the stimulatory effects of CRH on the POMC promoter. A schematic diagram of the proposed cross-talk mechanism between Shh and CRH pathways is given in Fig. 3
.
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We show here that Shh and components of its signal transduction pathway are expressed and play a functional role in the adult pituitary gland. We describe a new cross-talk mechanism between the signaling pathways of CRH and Shh and identify Gli1 as a new factor that regulates the biosynthesis of ACTH in response to CRH.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2138fje;
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P < 0,01 compared with Shh; #P < 0,01 compared with CRH.
