AMP-activated Protein Kinase (AMPK) Regulate Progesterone Receptor (PR) Transcriptional Activity

Adenosine Monophosphate-activated Protein Kinase (AMP-activated protein kinase, AMPK), a serine/threonine protein kinase, regulates cellular energy homeostasis. It protects the cell as a'fuel gauge'by switching off ATP-consuming pathways and switching on alternative pathways for ATP generation in response to energy depletion. AMPK not only directly phosphorylates and regulates the metabolic enzymes as its short-term effects, but also influences on the pattern of gene expression in a variety of circumstances as its long-term activation through phosphorylation of transcription factors. However, there is still much unkown about the cellular AMPK signaling network. Search for the interaction proteins of AMPK provides a way to understand the mechanisms and functions of AMPK.Based on yeast two hybrid assay, we identified 71 proteins interacted with AMPKβ1 subunit in the human universal cDNA library. Among these proteins, there are 30 enzymes including metabolic enzymes, kinases and SUMO protease,9 transcription factors or their coregulators,5 transport/cargo proteins,4 GTP-binding proteins,3 adaptors and some proteins involved in cell cycle, DNA repair and cell growth and/or maintainance. These results suggested that AMPK may play a key role not only in the metabolism but also in non-metabolism processes in the cellular functions.We selected one of these proteins related with transcription process and carried on further investigation. The ovarian steroid hormone progesterone is essential not only for normal female reproductive system, pregnancy and mammary development, but also for carcinoma in target tissues. The diverse effects of progesterone are mediated by the progesterone receptor (PR). The transcriptional ligand-dependent mechanism of receptor activation after binding of hormone to the ligand binding domain involves multiple steps, including a conformational change and dissociation from a multiprotein sequenestering complex consisting of protein chaperone molecules including heat shock proteins (hsp) 90 and other proteins. Progesterone receptors then undergo restructuring and dimerization. Activated receptors bind directly to specific progesterone response elements (PREs) or PRE-like sequences in the promoter regions of target genes. Recruiting coactivators to PR facilitates the assembly of the preinitiation complex at the promoter and increase rates of target gene transcription. The functions of PR are regulated not only by its ligands but also by modulators of various cell signaling pathways. However, whether energy state regulates PR activity is not clear.Using coimmunoprecipitation (CoIP) assays, we found association between PR and AMPKα1,α2 andβ1 subunits. It suggested that AMPK may regulate the PR signaling patyway. So, we investigated the effects of AMPK on the PR transcriptional activity then. Using the progesterone response element (PRE) drived luciferase reporter assay, we found that 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) and metformin, both of which are the pharmacological activator of AMPK, inhibited the PR pathway. The inhibitory effect by AICAR and metformin was reversed by Compound C, an inhibitor of AMPK. Down regulation of endogenous AMPK using small interfering RNA (siRNA) increases the PR activity. It suggested that AMPK is probably involved in the inhibition of the PR signaling pathway. Further results showed that the AMPK activated by AICAR altered the phospholation status of PR. AICAR-induced AMPK activation did not appreciably alter PR ligand-induced nuclear translocation, but blocked the transactivation by SRC3. AMPK did not changed the threshold concentration of progesterone, but reduced the highest level of transcriptional activity of PR induced by progesterone.In summary, through screening by yeast two hybrid, we identified many potential AMPK interacting proteins, involving in different kinds of processes in the cellular functions. In the further investigation, we confirmed AMPK interacted with PR in mammalian cells. We first found that activated AMPK altered the phosphorylation status of PR and inhibited its transcriptional activity. These findings suggest that AMPK have more roles in the cell signaling pathways, which may further our understanding of the ability of AMPK. Besides, the regulation of PR transcriptional activity by AMPK may provided valuable clues to understanding the relationship between progesterone pathway and energy status.