MTA1 and MTA3 Regulate HIF1a Expression in Hypoxia-Treated Human Trophoblast Cell Line HTR8/Svneo.

Hypoxia plays an important role in placental trophoblast differentiation and function during early pregnancy. Hypoxia-inducible factor 1 alpha (HIF1a) is known to regulate cellular adaption to hypoxic conditions. However, our current understanding of the role of HIF1a in trophoblast physiology is far from complete. Metastasis Associated Protein 1 and 3 (MTA1 and MTA3) are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a chromatin remodeling complex, and are highly expressed in term placental trophoblasts. However, the role of MTA1 and MTA3 in the hypoxic placental environment of early pregnancy is unknown. In the present study, we examined the association among MTA1, MTA3 and HIF1a expression under hypoxic conditions in trophoblasts both in vivo and in vitro. We first investigated the localization of MTA1 and MTA3 with HIF1a expression in the placental trophoblast of 1st trimester placenta via immunohistochemistry. Our data reveals that under physiologically hypoxic environment, MTA1 and MTA3 along with HIF1a are highly expressed by villous trophoblasts. Next, we investigated the effect of hypoxia on these genes in vitro using the first trimester-derived HTR8/SVneo cell line and observed up-regulation of MTA1 and MTA3 as well as HIF1a protein following hypoxia treatment. To investigate the direct effect of MTA1 and MTA3 upon HIF1a, we over-expressed MTA1 and MTA3 genes in HTR8/SVneo cells respectively and examined protein levels of HIF1a via Western blot as well as HIF1a target gene expression using a luciferase assay driven by a hypoxia-response element promoter (HRE-luciferase). We found that over-expressions of MTA1 and MTA3 up-regulate both HIF1a protein level and HRE-luciferase activity under hypoxic condition. In summary, both MTA1 and MTA3 are induced by hypoxia and up-regulate HIF1a expression and HIF1a target gene expression in trophoblasts. These data suggest that MTA1 and MTA3 play critical roles in trophoblast function and differentiation during early pregnancy. Central Uddin et al. (2013) Email: MNUDDIN@sw.org Med J Obstet Gynecol 1(3): 1017 (2013) 2/5 two subunits, HIF1a and HIF1b. HIF1a is regulated by O2 partial pressure and rapidly degraded under the normoxic condition with a half-life of less than 5 min. Hence, HIF1 activity is dependent mainly upon available amounts of HIF1a. It is known that HIF1a regulation occurs predominantly at the post-translation level via protein stabilization in response to O2 partial pressure. However, recent studies in cancer cells have shown that HIF1a is also responsive to post-translational modification, such as acetylation [8]. However, studies examining the regulatory mechanism of HIF1a protein stability within trophoblasts are limited. MTA1 and MTA3 are components of the Nucleosome Remodeling and Deacetylation complex (NuRD) which regulate protein acetylation (e.g. histone) via its de-acetylation activity. MTA1 and MTA3 are expressed in full term placenta [9], and have been previously shown to regulate genes implicated in trophoblast fusion and invasion [10]. However, the expression of MTA1 and MTA3 in the hypoxic placenta of early pregnancy and an examination into their potential role in hypoxia response and HIF1a regulation within trophoblasts has not been reported. Previous report has shown that, in cancer cells, overexpression of MTA1 up-regulates HIF1a protein level via adjusting its acetylation level [11]. Hence in this study, we investigated whether MTA1 and MTA3 regulate HIF1a in the placental trophoblasts of early pregnancy. Our results show that MTA1 and MTA3 are involved in the hypoxia response cascade through regulation of HIF1a protein level in trophoblasts. MaterIals and Methods Placental samples Immunohistochemistry (Ihc) De-identified formalin-fixed and parrafin wax embedded blocks of 9-week human placenta sections were obtained from Michigan State University’s Center for Women’s Health Research, Human Female Reproductive Tract Biorepository in accordance with appropriate institutional review. 4μM sections were dewaxed in xylene, rehydrated in a graded ethanol series and subjected to antigen unmasking with a high PH 9.0 buffer (Vector). Primary immunostaining with antibodies specific to MTA3 (Abcam 87275), MTA1 (Cell signaling 5647) and HIF1a (RD MTA1 Reverse: 5`GTCCTCGATGACGATGG-3`; MTA3 Forward: 5`-ATGGCGGCCAACATGTACCGGGT-3`; and MTA3 Reverse: 5`AGAATTTAAAAGCATCTTACA-3`) and then inserted into Lentivirus vector pLenti6-V5, downstream of CMV promoter (Invitrogen). After transfection of MTA1V5 and MTA3V5 vectors with packaging plasmids (Invitrogen) into 293 cells, according to manufacturer’s instructions. Lentivirus from the supernatant of culture media of 293 cells was collected and stored at -70°C until future use. HTR8/SVneo cells were infected by adding 100 μl lentivirus containing media per well to the cultured trophoblasts and passaged at least 5 times in the presence of 5ng/ml Blasticidin (approximately 1 month). HTR8/SVneo cells similarly infected with empty vector (pLenti-V5) were used as controls. Transgenic along with control cells were used for functional and biochemical analysis.