ECM-Dependent HIF Induction Directs Trophoblast Stem Cell Fate via LIMK1-Mediated Cytoskeletal Rearrangement

Abstract

The Hypoxia-inducible Factor (HIF) family of transcriptional regulators coordinates the expression of dozens of genes in response to oxygen deprivation. Mammalian development occurs in a hypoxic environment and HIF-null mice therefore die in utero due to multiple embryonic and placental defects. Mouse embryonic stem cells do not differentiate into placental cells; therefore, trophoblast stem cells (TSCs) are used to study mouse placental development. Consistent with a requirement for HIF activity during placental development in utero, TSCs derived from HIF-null mice exhibit severe differentiation defects and fail to form trophoblast giant cells (TGCs) in vitro. Interestingly, differentiating TSCs induce HIF activity independent of oxygen tension via unclear mechanisms. Here, we show that altering the extracellular matrix (ECM) composition upon which TSCs are cultured changes their differentiation potential from TGCs to multinucleated syncytiotropholasts (SynTs) and blocks oxygen-independent HIF induction. We further find that modulation of Mitogen Activated Protein Kinase Kinase-1/2 (MAP2K1/2, MEK-1/2) signaling by ECM composition is responsible for this effect. In the absence of ECM-dependent cues, hypoxia-signaling pathways activate this MAPK cascade to drive HIF induction and redirect TSC fate along the TGC lineage. In addition, we show that integrity of the microtubule and actin cytoskeleton is critical for TGC fate determination. HIF-2α ensures TSC cytoskeletal integrity and promotes invasive TGC formation by interacting with c-MYC to induce non-canonical expression of Lim domain kinase 1–an enzyme that regulates microtubule and actin stability, as well as cell invasion. Thus, we find that HIF can integrate positional and metabolic cues from within the TSC niche to regulate placental development by modulating the cellular cytoskeleton via non-canonical gene expression.

Three-dimensional cultures of trophoblast stem cells autonomously develop vascular-like spaces lined by trophoblast giant cells

“…Deletion of Hif1α/Hif2α and Arnt alters differentiation of trophoblast both in vivo and in vitro (Adelman et al, 2000;Cowden Dahl et al, 2005). In addition to regulation by oxygen, however, HIF activity in TS cells is also regulated by the extracellular matrix (Choi et al, 2013). Mutation in gap junction protein C Â 31 shifts fate from P-TGCs to S-TGCs and/or Ch-TGCs due to reduced of oxygen sensing ability (Koch et al, 2012).…”

“…The extracellular microenvironment of cultured mouse TS cells has been shown to affect the differentiation of syncytiotrophoblast versus TGCs. (Choi et al, 2013). The five TGC subtypes form in specific locations within the mouse placenta, suggesting that there may be location-specific cues.…”

“…Knockout mice for Hif1α, Hif2α and Arnt have shown that these factors also regulate trophoblast differentiation (Cowden Dahl et al, 2005). TS cells derived from Hif mutants also show defects in differentiation (Choi et al, 2013). Hif1α/2α double mutants and Arnt mutants show reduced expression of the P-TGCspecific gene Prl3d1 (Adelman et al, 2000;Cowden Dahl et al, 2005), though at the time of the analysis, markers of other TGC subtypes were not assessed.…”

Placenta: The Forgotten Organ

“…Along these lines, invasive TGCs in mice and EVTs in humans exhibit robust microtubule and actin cytoskeletons, whereas multinucleated SynTs in both species exhibit severely disrupted cytoskeletons (Choi et al 2003, Zhou et al 2014. Consistent with these observations, microtubule-or actin-disrupting agents direct TSC differentiation along the SynT lineage (Choi et al 2013). Caspase 8 activity, frequently implicated in apoptosis, aids the process of human SynT formation, cleaving α-fodrin, a component of the submembranous cytoskeleton (Huppertz et al 1999).…”

“…Feeder cells produce growth factors such as the TGFβ family member Nodal (Erlebacher et al 2004, Guzman-Ayala et al 2004) as well as extracellular matrix-dependent cues (Choi et al 2013) to help recreate the stem cell niche that maintains TSC proliferation and prevents their differentiation. In utero, this environment is maintained for approximately 3-4 days following implantation, as TSCs cannot be derived from mouse embryos past embryonic day (E) 6.5 (Uy et al 2002).…”