Involvement of a chromatin modifier in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury: Probably an indirect action via the regulation of NFκB/FasL circuitry

https://doi.org/10.1016/j.bbrc.2013.09.135Get rights and content

Highlights

  • MTA1 expression is upregulated in SCs upon MEHP treatment.

  • Knockdown of MTA1 in SCs impairs the MEHP-induced NFκB signaling activation.

  • Knockdown of MTA1 inhibits recruitment of NFκB onto FasL promoter in MEHP-treated SCs.

Abstract

The Fas/FasL signaling pathway, controlled by nuclear factor-κB (NFκB) at the transcriptional level, is critical for triggering germ cell apoptosis in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell (SC) injury, but the exact regulation mechanism remain unknown. Here, we discovered that expression level of Metastasis associated protein 1 (MTA1), a component of the Mi-2/nucleosome remodeling and deacetylase complex, was upregulated in SCs during the early recovery after MEHP exposure. This expression change was in line with the dynamic changes in germ cell apoptosis in response to MEHP treatment. Furthermore, a knockdown of MTA1 by RNAi in SCs was found to impair the MEHP-induced early activation of NFκB pathway and abolish the recruitment of NFκB onto FasL promoter, which consequently diminished the MEHP-triggered FasL induction. Considering that Fas/FasL is a well characterized apoptosis initiating signaling during SCs injury, our results point to a potential “switch on” effect of MTA1, which may govern the activation of NFκB/FasL cascade in MEHP-insulted SCs. Overall, the MTA1/NFκB/FasL circuit may serve as an important defensive/repairing mechanism to help to control the germ cell quality after SCs injury.

Introduction

Phthalic acid esters are widespread in the environment due to their use as plasticizers to produce plastic wraps, toys, and bags, and have been frequently shown to adversely affect spermatogenesis [1]. Among phthalic acid esters, di-(2-ethylhexyl) phthalate (DEHP) is widely dispersed throughout the environment due to its increased commercial use. Mono-(2-ethylhexyl) phthalate (MEHP), one of the active metabolites of DEHP, is a well known Sertoli cell (SC) toxicant [2]. The testicular toxicity is characterized with detachment and sloughing of spermatogenic cells due to increased germ cell apoptosis and atrophy in seminiferous tubules [3].

The FasL/Fas signaling pathway has been established to operate as an essential mechanism to regulate germ cell apoptosis in the testis as part of a physiologic pathway to match germ cell numbers to the SCs supportive capacity [4]. Recent studies indicate that the induction of FasL protein expression in germ cells by MEHP treatment is critical for initiating massive germ cell elimination by apoptosis after SCs injury [5]. MEHP-induced FasL expression requires the transcriptional regulation of transcription factors such as nuclear factor-κB (NFκB) as well as specificity protein-1 (Sp-1) [6]. However, the exact regulation of Sertoli cell NFκB/FasL expression in response to MEHP remains to be fully delineated.

Regulation of fundamental germ cell apoptosis demands dynamic coordinated participation of transcription factors and their coregulators at the target gene chromatin [7]. Metastasis associated protein 1 (MTA1), a component of the Mi-2/nucleosome remodeling and deacetylase complex, plays a central role in the regulation of divergent cellular pathways by associating and modifying the acetylation status of the target gene chromatin [8]. In testis, MTA1 is predominantly expressed in pachytene spermatocytes and weakly expressed in SCs [9], [10], [11]. Data from this lab have shown that MTA1 might operate as an indispensable modulator in the maintenance of the proper apoptotic balance of meiotic spermatocytes under certain pathologic conditions [12], [13]. In addition, SC-expressing MTA1 is crucial to maintain the germ cell nursery function and normal anchoring junction formation in SCs [14]. Although MTA1 has been linked intimately with spermatogenesis and is widely regarded as a potential master coregulator, the role of MTA1 in SCs injury remains unrecognized and delineated here. For the first time, we provide evidence that NFκB controlled FasL expression after MEHP exposure is governed by MTA1 originated from SCs. Our results reiterate the importance of paracrine interactions between SCs and germ cells during SCs injury.

Section snippets

Animal treatment

Adult male C57BL/6 mice, obtained from the Animal Research Center of our university, were given a single dose of MEHP (1 g/kg) (Sigma) by oral gavage. Control animals (n = 5) received a similar volume of vehicle (corn oil). Mice were then sacrificed at different time-points (n = 5 for each time-point) by CO2 inhalation. All procedures involving animals were approved by the local ethical committee.

Cell preparation and treatment

Sertoli cells (SCs) were prepared as described elsewhere [14]. SCs Cultures were hypotonically treated

MTA1 expression in mouse SCs is induced by MEHP in a time-dependent manner

Immunohistochemistry was firstly employed to evaluate the influence of MEHP on MTA1 expression level in adult mouse testis. Mice were given a single dose of MEHP by oral gavage and testicular tissues were then collected at different time-points after treatment. MTA1 staining in SCs was significantly increased from the postoperative 12 h onwards (black arrows in Fig. 1A). In contrast, positive signals in pachytene spermatocytes (pachy) remained relatively constant and began to decrease at

Discussion

Accumulated evidence establishes MTA1 to be a valid DNA-damage responsive protein with a master co-regulatory role in maintaining the optimum DNA-repair activity in mammalian cells [18]. Genotoxic stimulations such as hyperthermia [12], ionizing radiation [19], chemotherapy and oxidative stress [20] could significantly deregulate the expression level of MTA1. It is therefore a logical observation that this chromatin modifier was upregulated in response to MEHP treatment. Considering that MTA1

Acknowledgments

We are indebted to Miss Hui Wang (Department of Foreign Language, Fourth Military Medical University, China) for her careful assistance during the preparation of the manuscript. This work was supported by the Natural Science Foundation of China (NSFC) (31271248).

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These authors contributed equally to this work.

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