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TNF-α-mediated suppression of Leydig cell steroidogenesis involves DAX-1

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Abstract

Objective and design

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has an inhibitory role in gonadal functions particularly in the steroidogenesis of Leydig cells. A detailed understanding of the mechanisms by which TNF-α regulates testicular steroidogenesis will be helpful in the design of novel clinical interventions for the treatment and prevention of male reproductive disorders. Here, we report that TNF-α-mediated activation of DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is involved in the inhibition of Leydig cell steroidogenesis.

Materials and methods

Rat testis Leydig tumor cells (LC-540) were treated with TNF-α (10 ng/ml) for different time intervals. To elucidate the pathways of intracellular signal transduction that regulate DAX-1 expression, we utilized specific inhibitors. The siRNA transfection of DAX-1 into LC-540 cells was performed by electroporation. The mRNA and protein levels were determined by RT-PCR and Western blotting, respectively.

Results

We found that the mRNA and protein levels of DAX-1 were increased by threefold approximately in TNF-α-treated cells when compared to controls. Staurosporine, JNK inhibitor SP600125 and ERK inhibitor PD98059 significantly decreased DAX-1 expression in TNF-α-treated Leydig cells when compared to their respective controls. Further, a siRNA-mediated knockdown of DAX-1 restores the expression of steroidogenic proteins in TNF-α-treated Leydig cells.

Conclusions

These findings provide valuable information that TNF-α activates DAX-1 through JNK/ERK MAP kinase pathway which regulates the expression of steroidogenic enzyme genes in Leydig cells.

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Acknowledgments

The authors gratefully acknowledge Indian Council of Medical Research (ICMR), New Delhi, India for the financial assistance. The infrastructure provided by DST-FIST is also gratefully acknowledged.

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Authors and Affiliations

  1. Department of Biochemistry, Bharathidasan University, Tiruchirappalli, 620 024, India

    Mohanraj Sadasivam, Balamurugan Ramatchandirin, Sivasangari Balakrishnan & Chidambaram Prahalathan

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  1. Mohanraj Sadasivam
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  2. Balamurugan Ramatchandirin
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  3. Sivasangari Balakrishnan
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  4. Chidambaram Prahalathan
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Corresponding author

Correspondence to Chidambaram Prahalathan.

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Responsible Editor: Andrew Roberts.

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11_2015_835_MOESM1_ESM.tif

Supplementary material 1 LC-540 cell viability is affected by TNF-α in a dose- a and time- b dependent manner. Values represent mean ± SD for 3 independent experiments (n=3). Values are statistically significant at *** P < 0.001, **P < 0.01 and * P < 0.05 (TIFF 153 kb)

11_2015_835_MOESM2_ESM.tif

Supplementary material 2 Effect of NF-κB inhibitor on DAX-1 protein levels in LC-540 Leydig cells cells. LC-540 Leydig cells were pretreated with BAY11-7082 (10µM) for 60min and then treated with or without TNF-α (10ng/ml) and used for the analysis of DAX-1 protein expressions. β-actin levels were utilized as internal control. Values represent mean ± SD for 3 independent experiments (n=3). Values are statistically significant at *** P < 0.001 (TIFF 150 kb)

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Sadasivam, M., Ramatchandirin, B., Balakrishnan, S. et al. TNF-α-mediated suppression of Leydig cell steroidogenesis involves DAX-1. Inflamm. Res. 64, 549–556 (2015). https://doi.org/10.1007/s00011-015-0835-8

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  • DOI: https://doi.org/10.1007/s00011-015-0835-8

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