Figure 1.
HIF integrates ECM cues and Oxygen Levels to Direct TSC Fate.
(A–D) Immunofluorescence microscopy of undifferentiated control TSCs cultured on CELLstart™ with anti-CDX2 and EOMES antibodies (blue = DAPI, red = CDX2, green = Eomes). (E, G) Phase contrast microscopy of control TSCs maintained on CELLstart™ following differentiation for 7 days under normoxic (21% O2) or hypoxic (2% O2) conditions. (F, H) Immunofluoresce microscopy of control TSCs maintained on CELLstart™ following differentiation for 7 days under normoxic (21% O2) or hypoxic (2% O2) conditions with an anti-HOPX1 (red) antibody (blue = DAPI). (I) Quantitative RT-PCR analysis of Pl. I, Pl.II, Ctsq, Plf, Tfeb, SynA and SynB gene expression in wild-type (+/+) TSCs differentiated for 7 days following culture on CELLstart™ or on TC plastic in Fib-CM, compared with Arnt−/− (−/−) TSCs differentiated following culture on TC plastic in Fib-CM. p values <0.05 versus wild-type Fib-CM indicated by an asterisk. (J) Immunoblot of HIF-1α and -2α protein levels in whole cell lysates of wild-type TSCs differentiated for 7 days following culture on CELLstart at 21%O2 or 2% O2. (K) Quantitative RT-PCR analysis of Pl-1, Pl-2, Plf, Tfeb, SynA and SynB expression in Vhlh+/+ and Vhlh−/− TSCs differentiated following culture on CELLstart™. p values <0.05 versus wild-type indicated by an asterisk.
Figure 2.
β3-Integrin (CD61) is downregulated in TSCs following culture on CELLstart™.
Immunofluoresce microscopy of wild-type TSCs maintained on TC plastic in Fib-CM (A) or on CELLstart™ (B) in the presence of FGF4 and heparin (Undiff.) or following differentiation (Diff., C and D) using an anti-CD61 antibody (green) (magnification 630X).
Figure 3.
ECM- or oxygen-dependent HIF-α subunit stabilization and TGC formation are dependent on MAP2K1/2 activity.
(A) Immunoblot of whole cell lysates obtained from TSCs differentiated in 2% or 21% O2, with and without U0126, following culture on CELLstart™, for HIF-1α, -2α or α-Tubulin. (B) Immunoblot of whole cell lysates obtained from differentiated wild-type TSCs following culture on TC plastic in Fib-CM with and without U0126 with a HIF-1α antibody. (C) (D) Immunofluorescence microscopy of TSCs maintained on CELLstart™ following differentiation for 7 days under hypoxic conditions without and with U0126 (10 uM) using anti β-Catenin antibodies (green) (blue = Dapi). (E) Quantitaive RT-PCR analysis of Plf, Pl-I, Ctsq, 4311, Mash2, Tfeb and SynA expression following differentiation of wild-type TSCs cultured on CELLstart™ under hypoxic conditions without and with U0126. p values <0.05 versus drug free control indicated by an asterisk. (F) Immunofluorescence microscopy using anti HA (red) and β-Catenin (green) antibodies of control TSCs differentiated following culture on CELLstart™ under 21% O2 following transient tranfection with constitutively active HA:MAP2K1 or under (G) 2% O2 following transient transfection with dominant negative HA:MAP2K1. (H, I) Immunofluorescence microscopy of wild-type TSCs differentiated following culture on TC plastic in Fib-CM in 21% O2 with and without U0126 with antibodies for HDAC2 (red) and E-Cadherin (green). (J) Northern blot analysis of lineage specific marker gene expression in wild-type TSCs maintained on TC plastic in Fib-CM and differentiated with and without U0126, compared with differentiated Arnt−/− TSCs.
Figure 4.
MAP2K1/2 inhibition and cytoskeletal rearrangement in differentiating HIF-null TSCs.
Immunofluorescence microscopy of terminally differentiated wild-type (+/+) TGCs (A) and Hif-1/2α−/− (−/−) SynTs (B) with an anti α-Tubulin (red), or p-MAPK3/1 (green) antibody (arrows = microtubules, arrowheads = pMAPK3/1). (C) Confocal microscopy imaging of polymerized actin via FITC-phalloidin staining (green) or (D) α -Tubulin (red) in terminally differentiated control TGCs (dashed line indicates approximate location of nucleus). (E) Confocal microscopy imaging of polymerized actin via FITC-phalloidin staining (green) or (F) α-Tubulin (red) in terminally differentiated HIF-null SynTs. (G) Differentiation of control TSCs in the presence of Taxol (G) or (H) Cytochalasin B (CB) promoted the formation of multinucleated cells (arrowheads) following culture on TC plastic in Fib-CM. α-Tubulin (red) and E-Cadherin (green).
Figure 5.
HIF-dependent LIMK1 expression promotes TGC formation in TSCs.
Immunofluorescence microscopy of terminally differentiated control TGCs (A) and Hif-1/2α−/− SynTs (B) with a β-Catenin (red) and LIMK1 (green) antibody (arrows = perinunclear LIMK1 staining). Immunofluorescence microscopy of terminally differentiated control TGCs (C) and Hif-1/2α−/− SynTs (D) with a β-Catenin (green) and p-Cofilin (red) antibody (arrows = perinunclear p-Cof staining, arrowheads = cofilin rods). (E, F) Two representative images of TGC formation (arrows) following transient myc-LIMK1 expression in Hif-1/2 −/− TSCs while untransfected cells primarily form SynTs (arrowheads) (red = myc-LIMK1, green = β-catenin). (G) Quantification of the percentage of LIMK1 transfected HIF-null TSCs differentiated into TGCs vs. SynTs. (H) Immunoblot analysis of LIMK1 levels in differentiated wild-type (+/+) TSCs without and with U0126 (U0). Integrated densitometry confirmed the decreased expression of LIMK1, relative to total Cofilin, in control TSCs differentiated in the presence of U0126.
Figure 6.
Canonical vs non-canonical HIF target gene-expression in TS cells.
(A) Electrophoretic mobility shift assay (EMSA) of differentiated control TGC nuclear extracts with and without 2 different anti-HIF-1α antibodies (H1) or a HIF-2α antibody (H2) (“supershift” SS, NS, non-specific complexes.) (B) Schematic representation of full-length HIF-1α and HIF-2α, as well as versions lacking their DNA binding basic (b) domains (HLH, Helix-loop-helix, PAS, Per-Arnt-Sim, ODDD, oxygen-dependent degradation domain). (C) Immunoblot detection of stable HA-epitope tagged HIF-1α, HIF-1αΔb, HIF-2α and HIF-2αΔb protein, as well as respective target gene protein products in Hif-1/2α−/− TSCs. (D) Integrated densitometric quantification of HIF target gene protein products relative to Actin expression in each respective cell line.
Figure 7.
Canonical target gene-independent HIF-2 activity drives LIMK1 expression in TS cells via c-MYC interaction.
(A) Immunoblot analysis of LIMK1 protein levels in Hif-1/2α−/− TSCs stably reconstituted with full length HIF-1α or -2α, as well as versions lacking their basic domains. (B) Immunoblot analysis of LIMK1 and LIMK2 expression in control (+), Hif-1/2α−/− (Hif−/−), and HIF-2α and HIF-2αΔb reconstituted Hif-1/2α−/− TSCs. Integrated densitometric analysis confirmed that both HIF-2α, as well as HIF-2αΔb, restored LIMK1 expression to control levels in Hif-1/2α −/− TSCs. (C) Immunoprecipitation with an anti-HA antibody of HA-tagged HIF-2αΔb followed by immunoblot with anti-HA, c-MYC, β-Catenin, α-Tubulin and GFP antibodies. (D) Schematic representation of E-box element identified within the Limk1 promoter. Chromatin immunoprecipitation (ChIP) analysis indicated specific binding of c-MYC and HA-tagged HIF-2αΔb to this element. (E) Immunoblot analysis of LIMK1 protein levels in HIF-2αΔb expressing Hif-1/2α−/− TSCs without (-) or with (+) c-MYC inhibitor. Integrated densitometric analysis confirmed reduced expression of LIMK1 relative to α-Tubulin in drug treated cells. (F) Schematic representation of HIF-2α interacting with MYC:MAX heterodimers at the Limk1 promoter.
Figure 8.
Non-canonical HIF-2-dependent LIMK1 expression promotes TGC formation.
Immunofluorescence microscopy of HIF-2αΔb expressing Hif-1/2α−/− TSCs differentiated following culture on TC plastic in Fib-CM (A) or on CELLstart™ (B) (green = β-Cat, red = HA). (C) qRT-PCR-based comparison of expression levels of the TGC (PLF, Pl-2, Ctsq), spongiotrophoblast (Mash2, 4311) and SynT (Tfeb, SynA) markers in Hif-1/2α−/− and HIF-2αΔb reconstituted HIF-null (Hif−/−:2αΔb) TSCs differentiated for 7 days following culture on TC plastic in Fib-CM. p values <0.05 versus HIF-null indicated by an asterisk. (D) Immunofluorescence microscopy of HIF-2αΔb expressing Hif-1/2α−/− TSCs differentiated following culture on TC plastic in Fib-CM in the presence of Cytochalasin B or (E) the LIMK inhibitor (BMS-5 10 uM)(green = β-Cat). (F) qRT-PCR-based comparison of expression levels of the TGC (PLF, Pl-1, Pl-2, Ctsq), spongiotrophoblast (Mash2, 4311) and SynT (Tfeb, SynA) markers in HIF-2αΔb reconstituted HIF-null (Hif−/−:2αΔb) TSCs differentiated for 7 days following culture on TC plastic in Fib-CM without and with the actin cytoskeleton disrupting agent cytochalasin B (Cyto B). p values <0.05 versus drug free control indicated by an asterisk. (G) Immunofluorescence microscopy of HIF-2αΔb expressing Hif-1/2α−/− TSCs differentiated following culture on CellStart™ in the presence of Cytochalasin B or (H) the LIMK inhibitor (BMS-5 10 uM)(green = β-Cat).
Figure 9.
Model of HIF-dependent integration of positional and metabolic cues in the TSC niche.
ECM composition regulates HIF stabilization likely downstream of cell surface integrin ligation via MAP2K1/2 activation. Inside-out integrin signaling mechanisms may also be operative. Oxygen sensing and signaling pathways intersect with this signaling cascade to stabilize HIF, when ECM-dependent cues are absent. Stabilized HIF can act via canonical and non-canonical target genes. Non-canonical HIF-2, by interacting with MYC:MAX heterodimers, bind the Limk1 promoter to activate its expression. LIMK1 promotes microtubule and actin stability, critical for TGC formation, and thereby prevents SynT formation. HIF, therefore, can integrate divergent environmental inputs from within the placenta to regulate cell fate via non-canonical gene expression.