Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics

Abstract

Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increases cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B and PDE4C as well as PDE8A and PDE8B are expressed in rodent Leydig cells and that combined inhibition of PDE4 and PDE8 leads to dramatically increased steroid biosynthesis. Here we investigated the effect of PDE4 and PDE8 inhibition on the molecular mechanisms of cAMP actions in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE4 family specific inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180min. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests that cAMP, PDE4 and PDE8 coordinate steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that may be regulated to assure timely and sufficient testosterone secretion in response to LH.

Keywords: Leydig cells; Phosphodiesterase; Phosphorylation; Proteomics; SILAC; Steroidogenesis.

Figure 1

A) Experimental setup for the triple SILAC labeling experiments in MA10 cells comparing single PDE4 (Rolipram, Rol) or PDE8 (PF-04957325, PF) inhibitor treatment with PDE4 and 8 inhibitor combination treatment for 1 h and the setup of the time series experiment with PDE4 and 8 inhibitor treatment for 0, 1, 15, 60 and 180 minutes. B) Phosphoproteomics workflow according to which MA10 cell samples were processed and analyzed.

Figure 2

A) Plot of the log10 protein intensity and the protein intensity rank (n = 8234). Abundance of a protein was estimated to increase in parallel with its intensity. The proteins were divided into quartiles of intensity and a GO term enrichment was conducted for each of the quartiles using the GOrilla web application. Shown are examples for enriched terms for GOBP, GOMF and GOCC. B) Venn diagram comparing the proteins identified in the background proteome and the phosphoproteome. C) Venn diagram comparing the proteins quantified in the background proteome and the phosphoproteome. D) Comparison of the number of identified phosphosites, quantified phosphosites and regulated phosphosites in the dataset. D) Plot of all log2 SILAC ratios in the phosphoproteomics dataset. 90% of unique phospho sites showed a log2 SILAC ratio within a range of >0.6±0.17 and <0.53±0.07 (mean±SD). Phosphosites showing log2 SILAC ratios outside this data interval were considered potentially regulated by PDE inhibitor stimulation.

Figure 3

A) Temporal clustering of phosphorylation changes quantified in the phosphoproteomics PDE4 and 8 inhibition time series experiment. Clustering was performed using the Mfuzz package in R with six clusters, only considering sites that are quantified in all experiments and that have a minimum abs log2 SILAC ratio of 0.2. B) and C) IceLogos created from the ±7 aa sequence window of significantly up- or down-regulated phosphorylation sites and pie charts of predicted upstream kinases targeting these phosphorylation sites. Sites were considered up- or down-regulated if increased or decreased in the 1 h PDE inhibition experiment or if they could be clustered to the corresponding temporal clusters (upregulated = cluster 2, 3, 4 and down-regulated = cluster 1, 5, 6) for sites derived from the time series experiment. Sequence logos were created using IceLogo 1.2 with the entire Mus musculus genome as background data set. Upstream kinases were predicted with the NetPhorest web tool (http://www.netphorest.info/index.shtml) using mouse protein sequences and human kinase substrate consensus sequences. Only predicted kinases were considered that showed a NetPhorest score of >0.1.

Figure 4

A) Pie chart showing predicted upstream kinases (NetPhorest) for phosphosites specifically regulated by PDE8. B) Venn diagram showing the overlap between PDE8 and PDE4 and 8 inhibition-regulated phosphorylation sites.

Figure 5

Regulated phosphoproteins after PDE4 and 8 inhibition that could be functionally associated with specific cellular processes. Only proteins associated with a given process by i) being tagged with a corresponding GOBP term and ii) for which confirmatory literature reports exist linking these genes to the process are considered. For simplicity, individual biological processes regulated are shown in separate tiles.