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Cigarette smoke-induced cell cycle arrest in spermatocytes [GC-2spd(ts)] is mediated through crosstalk between Ahr–Nrf2 pathway and MAPK signaling Free
Prabagaran Esakky1,2, Deborah A. Hansen1, Andrea M. Drury1,2, and Kelle H. Moley2,*
1Research, Department of Veterans Affairs Medical Center, St. Louis, MO 63106, USA
2Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO 63110, USA *Correspondence to:Kelle H. Moley; E-mail: moleyk@wustl.edu
J Mol Cell Biol, Volume 7, Issue 1, February 2015, 73-87,  https://doi.org/10.1093/jmcb/mju049
Keyword: spermatocytes, Ahr, Nrf2, CSC, ATF3, E2F4, p38 MAPK, and ERK

Our earlier studies have demonstrated that the cigarette smoke in the form of cigarette smoke condensate (CSC) causes growth arrest of a mouse spermatocyte cell line [GC-2spd(ts)] through activation of the AHR–NRF2 pathway. The present study demonstrates the CSC-activated p38 and ERK MAPK signaling in GC-2spd(ts) via arylhydrocarbon receptor (AHR). Pharmacological inhibition by using AHR-antagonist, or p38 MAPK and ERK (MEK1) inhibitors significantly abrogates CSC-induced growth arrest by AHR and MAPK inactivation. QRT-PCR, western blot, and immunofluorescence of Ahr-target of Nrf2, and stress-inducible growth suppressive Atf3 and E2f4 following treatments indicate a crosstalk among these pathways. Regulation of Atf3 by Nrf2 and Ahr through RNA interference suggests the existence of a cross-regulatory loop between the targets. CSC induction of E2f4 via Atf3 and its regulation by pharmacological inhibitors reveal a possible regulatory mechanism of growth inhibitory CSC. SiRNA silencing of Ahr, Nrf2, Atf3, and E2f4 genes and downregulation of cyclins by CSC corroborate the growth inhibitory effect of cigarette smoke. Thus, the data obtained suggest that the CSC-mediated MAPKs and AHR–NRF2 crosstalks lay the molecular basis for the growth arrest and cell death of spermatocytes.