Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC1

Abstract

Our previous studies showed that the prototypical testicular toxic phthalate monoester, mono-(2-ethylhexyl) phthalate (MEHP), suppresses Sertoli cell TIMP2 levels and allows for the activation of MMP2 in seminiferous epithelium. Activation of MMP2 is important for triggering germ cell apoptosis and instigating germ cell detachment from Sertoli cells. These novel findings led us to examine the transcriptional regulation of the Timp2 gene that accounts for the decrease in Sertoli cell TIMP2 levels following MEHP exposure. Sequential deletion of the Timp2 5'-upstream activating sequence (1200 bp) was used to survey transcriptional activation in the Timp2 promoter region in response to MEHP. Results indicate that under control conditions in rat Sertoli cells, CCAAT enhancer-binding protein alpha (CEBPA) acts as a transactivator to initiate Timp2 gene transcription, and its action is deactivated by exposure to MEHP. By contrast, MYC protein acts as an inhibitor of Timp2 gene transcription, and its activity is increased after MEHP treatment. Addition of follicle-stimulating hormone (FSH) to cells causes translocation of CEBPA into the Sertoli cell nucleus and rescues MEHP-suppressed TIMP2 levels. Down-regulation of TIMP2 expression by MEHP exposure is blocked by forskolin (a cAMP-elevating agent), suggesting that the decrease in Sertoli cell TIMP2 expression following MEHP exposure is cAMP-dependent. Taken together, these data indicate that MEHP both disrupts the FSH-stimulated cAMP signaling pathway and activates the inhibitory signaling mediated by MYC protein, to ultimately account for the cellular mechanism underlying the decreased expression of TIMP2 in Sertoli cells.

Dissecting the Phthalate-Induced Sertoli Cell Injury: The Fragile Balance of Proteases and Their Inhibitors1

“…Indeed, it is now assumed that the concomitant transit of preleptotene spermatocytes across the BTB and the release of mature spermatozoa that are permitted by the restructuring of BSTB and apical ES, respectively, are coordinated events involving a local regulatory axis [31]. Supporting this view are the observations that the modification of expression of occludin and the appearance of gaps between Sertoli cells follow MEHP treatment as described in the study of Yao et al [8] and as shown by the dramatic decrease of transepithelial electrical resistance in Sertoli cell cultures treated with MEHP [21]; both studies address the question of the BTB permeability. Because TNF induces COL4A3, MMP9, and TIMP1, but not MMP2, during BTB deassembly [4], it would be interesting to dissect how MEHP can induce MMP2, but not MMP9, and can decrease both TIMP1 and TIMP2 while inducing TNF processing into its soluble form.…”

“…The study by Yao et al [8] represents a significant step forward in our understanding of the molecular events in the testis triggered by MEHP exposure. These findings are of particular importance at this time, given the growing concern about the role played by environmental toxicants on male reproductive function.…”

“…The approach taken to understand the range of molecular mechanisms following phthalate exposure, from transcriptional regulations to posttranslational modifications, can serve as a model for deciphering the actions of other reproductive toxicants. [8], by activating the expression of the transcriptional repressor MYC and by inhibiting the FSH-mediated nuclear translocation of the transcriptional activator CEBPA. The decrease of Timp2 expression concurs to MMP2 activation that itself contributes to TNF processing into its soluble form.…”

“…The MMP2/TIMP2 homeostasis is fragile and was previously reported to be sensitive to phthalate [7]. In the current issue of Biology of Reproduction, Yao et al [8] explore the transcriptional mechanism of Timp2 downregulation that follows phthalate exposure.…”

Implications of Sertoli cell induced germ cell apoptosis to testicular pathology

“…34 This allows for the activation of matrix metalloproteinase 2 (MMP2) in the adluminal space and the consequent production of a soluble form tumor necrosis factor-a (sTNFa), which ultimately induces the increased expression of FasL on Sertoli cells. [34][35][36] Although apoptosis typically does not induce inflammation, recent reports indicate that direct injury to Sertoli cells can change the normal immune privilege environment of the testis (disruption of the blood testis barrier, infiltration of macrophages). 37,38 Therefore, Sertoli cell injury can induce a variety of changes in the seminiferous epithelium including the induction of germ cell apoptosis and altered immune environment.…”