Inhibition of methylation stimulates POMC expression and ACTH secretion in human pituitary ACTH-secreting tumors
M. Cassarino1, A. Sesta1, L. Pagliardini1, M. Losa3, G. Lasio4, F. Cavagnini1 & F. Pecori Giraldi1,2
DNA methylation has recently been shown to play a role in postnatal as well as developmental gene regulation. Transfer and removal of methyl groups from CpG islands in the promoter region of specific genes has been associated with gene silencing or activation, respectively. POMC is known to present several methylation sites and demethylation of these sites has been shown to be linked to POMC expression by neuroendocrine tumors outside the pituitary.
Aim: Of the present study was to evaluate whether inhibition of methylation by DNA-metyltransferases (DNMTs) modulates POMC expression and ACTH secretion by pituitary corticotroph tumors.
Methods: Thirteen human pituitary ACTH-secreting tumors were collected at surgery and established in culture. Cells were incubated with 1 μM Aza-2′-deoxycytidine (AZA), an inhibitor of DNMTs, with or without 10 nM CRH. After 24 and 48 h, medium was collected for measurement of ACTH by IRMA and RNA extracted for evaluation of POMC expression by real-time PCR. Data was normalized to control wells (DMEM and BSA alone) and analyzed by Wilcoxons test.
Results: Incubation with AZA for 24 h increased POMC expression and ACTH secretion compared to control wells; lesser effects were observed after 48 h incubation. AZA did not appear to exert an additional stimulatory effect on POMC and ACTH stimulated by CRH.
Conclusions: The present study shows that inhibition of methylation increases POMC gene expression and ACTH secretion in human pituitary ACTH-secreting tumors. This can be taken to indicate that methylation plays a role in the regulation of gene expression in tumoral corticotrophs and paves the way to further study on epigenetic changes in Cushings disease.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
|ACTH (% control)||24 h||48 h|
|10 nM CRH||690.4±227.5*||525.9±154.4*|
|1 μM AZA||164.9±32.8*||101.3±6.5|
|POMC vs βGLUC|
|10 nM CRH||1.43±0.15*||2.12±0.43*|
|1 μM AZA||1.46±0.13*||1.43±0.26*|
|*P<0.05 vs control.|