Differential protein expression profile in the hypothalamic GT1-7 cell line after exposure to anabolic androgenic steroids

<div><p>The abuse of anabolic androgenic steroids (AAS) has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG) axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH). In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 μM). These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH <i>in vivo</i>. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.</p></div

Cellular functions and locations of differentially expressed proteins in GT1-7 cells after AAS exposure.

<p><b>(A)</b> A total of 8 biological processes with their respective cellular locations <b>(B)</b> were categorized from 17 different proteins identified by mass spectrometry. Data were obtained from Uniprot database (<a href="http://www.uniprot.org" target="_blank">http://www.uniprot.org</a>) and shown as percentages from the total number of proteins.</p

Validation of differentially expressed proteins in GT1-7 cells after AAS exposure.

<p><b>(A-E)</b> Representative Western blots of identified proteins from protein extracts of AAS or vehicle-treated cells. <b>(F-J)</b> Densitometry analysis of each protein normalized to β-actin represents the relative protein expression values for <b>(A)</b> Glutathione S-transferase Mu 1 (GSTM1), <b>(B)</b> Glyceraldehyde 3-phosphate dehydrogenase (G3P/GAPDH), <b>(C)</b> Enhancer of rudimentary homolog (ERH), <b>(D)</b> Phosphatidylethanolamine-binding protein 1 (PEBP1), <b>(E)</b> Protein disulfide-isomerase A6/Endoplasmic Reticulum Protein (ERP/PDIA6). Error bars represent standard error of the mean. *p≤0.05, **p≤0.01, unpaired t-test. n = three replicates of three independent experiments for each group.</p

Protein expression of steroids receptors and signaling pathway proteins in GT1-7 cells after AAS exposure.

<p><b>(A-E)</b> Representative Western blots of AR, ER, GnRH, p-ERK and p-p38 from protein extracts of AAS or vehicle-treated cells. <b>(F-J)</b> Densitometry analysis of each protein normalized to β-actin represents the relative protein expression values for <b>(A)</b> Androgen receptor (AR), <b>(B)</b> Estrogen receptor (ER), <b>(C)</b> GnRH, <b>(D)</b> Phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204), and <b>(E)</b> Phospho-p38 MAPK (p-p38), Error bars represent standard error of the mean. *p≤0.05, **p≤0.01, unpaired t-test. n = three replicates of three (AR and ER) or four (GnRH, p-ERK and p-p38) independent experiments for each group.</p

Overexpressed proteins in GT1-7 cells after AAS exposure.

<p>Overexpressed proteins in GT1-7 cells after AAS exposure.</p

Representative images of 2D-DIGE analytical gel and 3D view of protein spots in vehicle and AAS-treated GT1-7 cells.

<p><b>(A)</b> Twenty-three (23) protein spots were identified as differentially expressed in GT1-7 cells that were exposed to the AAS, 17α-meT (1μM). Protein samples were labeled with Cy5 (red), whereas those from the internal standard were labeled with Cy3 (green). After merging, protein spots exhibiting no changes appear yellow in DIGE images, underexpressed proteins appear in green, and overexpressed proteins are in red. White numbers and arrows correspond to the identified proteins in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180409#pone.0180409.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180409#pone.0180409.t002" target="_blank">2</a>. (<b>B-C)</b> Panels depict the protein spots’ abundance of <b>(B)</b> overexpressed and <b>(C)</b> underexpressed protein spots analyzed by DeCyder software. Purple lines encircle peaks representing protein spots from AAS-treated samples (left panels) or control vehicle (center panels). Protein abundance was calculated from normalized spot volume, standardized against the in-gel standard of each gel (right panels).</p

Network of proteins modulated by AAS in GT1-7 cells.

<p>Ingenuity Pathway Analysis (IPA) identified a major protein network associated with drug metabolism, glutathione depletion, protein synthesis, immunological disease and endocrine function. Red and green symbols indicate overexpressed and underexpressed proteins, respectively. GSTM2 is represented as GSTM5 in the IPA knowledge base. Direct interactions are represented as solid lines, whereas indirect interactions appear as dotted lines.</p