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Cooperative interaction between GATA5 and NF-ATc regulates endothelial-endocardial differentiation of cardiogenic cells

Laboratoire de Développement et Différenciation Cardiaques, Institut de Recherches Cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, Québec H2W 1R7, and Département de Pharmacologie, Université de Montréal, Canada

View larger version (82K): [in a new window]   Fig. 1. Characterization of the TC-13 cells. (A) When grown on matrigels, TC13 cells form row-like structures reminiscent of angiogenesis in vitro (–VEGF). When treated with an antibody against VEGF (+VEGF), row formation is inhibited and the cells stay rounded. (B) GATA5 transcripts are present only in differentiated cells. Northern blot analyses using 20 µg of total RNA isolated from undifferentiated (–RA) or differentiated (+RA) TC13 cells were used to detect Gata4 or Gata5 mRNA as described in Materials and Methods. (C) Identification of GATA binding activity. Gel Shift assays were carried out using 5 µg of nuclear extracts and a probe corresponding to the –90 bp GATA element of the BNP promoter as detailed in Materials and Methods. Note that GATA4-containing complexes have a higher mobility than GATA5 complexes. The GATA4 antibody totally supershifted the GATA binding in the undifferentiated cell extracts. GATA5 binding was present only in extracts from RA-treated cells and it was blocked by the GATA5 antibody. GATA4 binding was still detected after RA treatment but GATA5 represented the majority of GATA binding. (D) Control Oct1/2 binding using the same extracts as C. (E) Immunocytochemical staining of untreated (–RA) and treated (+RA) TC13 cells. Cells were fixed in methanol and incubation with the different antibodies was carried out overnight at 4°C as described in Materials and Methods. Staining was revealed by an FITC-avidin D conjugate antibody. Green fluorescent nuclear staining for GATA proteins and cytoplasmic labeling for Von Willebrand factor are observed. Note that only endothelial cells (elongated shape) are positive for GATA5 and Von Willebrand factor.

View larger version (132K): [in a new window]   Fig. 2. Endocardial expression of GATA4 and GATA5 in the developing heart. Immunocytochemical staining of staged mouse embryo sections with anti-GATA4 and -GATA5 polyclonal antibodies. (A) Low magnification of E10.5 stained embryos reveals abundant expression of GATA4 in atrial (A) and ventricular (V) myocytes, in endocardial cells, and in some cells of the endocardial cushion (EC). GATA5 labeling is restricted to endocardial (E) and endocardial cushion cells both in the atrio-ventricular cushion and in the truncus arteriosis (TA), which will give rise to the outflow tract. Staining is absent in the myocardium (M). (B) Expression of GATA5 is transient in endocardial cells. Note how GATA5 staining is undetectable at E12.5 whereas GATA4 is still present both in endocardial and myocardial cells. Counterstaining with Eosin and Hematoxylin.

View larger version (57K): [in a new window]   Fig. 3. Endocardial identity of differentiated TC13 cells. (A) Semi-quantitative RT-PCR analysis was carried out on total RNA isolated from undifferentiated (–RA) and differentiated (+RA) cells following 4 days of RA treatment, as well as from hearts (He) of neonatal Sprague Dawley rats. Tubulin transcript amplification was used as control. The oligonucleotides and conditions used are listed in Table 1. The results shown are from one representative experiment. (B) Immunocytochemical detection of the Cx37 and Cx40 gap-junction proteins. Note the specific green fluorescent labeling of the cell membrane with the anti-Cx37 antibody but not with the anti-Cx40 antibody. RNA analysis also confirmed the presence of Cx37 but not Cx40 transcripts in these cells (not shown). Nuclei are stained with the Hoechst dye.

View larger version (68K): [in a new window]   Fig. 4. Sequential expression of endothelial (A) and mesodermal genes (B). RT-PCR analysis was carried out on total RNA isolated at different stages of TC13 cell differentiation. Each amplification was carried out using the appropriate amount of cDNA as determined from a dose-response curve. For PCR conditions, please refer to Table 1. Amplified products were resolved on 1.2% agarose gels and visualized under UV light. (C) Gel shift analysis of NF-AT proteins in extracts from TC13 cells treated with RA for 0 (–) or 24 (+) hours. Specific complexes corresponding to NF-ATp and NF-ATc (Timmerman et al., 1997) are displaced by 100-fold excess of cold probe (competitor). Note how NF-ATc complexes are increased in RA-treated extracts. N.S, nonspecific.

View larger version (60K): [in a new window]   Fig. 5. Inhibition of endocardial differentiation in GATA5 antisense transfectants. (A) Schematic representation of the GATA5 antisense construct. The cDNA fragment spanned from 850 bp to 1200 bp where 1 is the A of the initiating methionine. (B) RT-PCR analysis was conducted on RNA isolated from two different TC13 clones expressing antisense GATA5 (AS5) as well as TC13 cells transfected with the control pCDNA3 vector. The GATA5 primers used detect only the endogenous transcripts. Note how the antisense inhibits accumulation of GATA5 transcripts following RA treatment. (C) Western blot analysis confirms that antisense clones do not express GATA5 protein. GATA4 levels are only slightly decreased in the GATA5 antisense but are further decreased in response to RA, as in control cells. 20 µg of nuclear extracts were resolved on a 15% SDS-PAGE and blotted on a nylon membrane with the respective antibodies. (D) GATA5-deficient TC13 cells do not elongate and remain negative for Von Willebrand factor staining even after 5 days of treatment with RA. No arrest in cell proliferation was observed in these cells compared to control cells. The data shown are from two independent clones, AS5(1) and AS5(2). (E) RT-PCR analyses show that terminal endocardial markers (EPAS-1, ET-1, Tn-X, ErbB3) are not induced in the antisense GATA5 transfectants after 4 days of RA treatment. (F) Gel shift analysis using extracts from RA-treated GATA5 antisense transfectants (AS) or RA-treated control (Ctl) in presence of cyclosporine A (CsA). NF-AT binding is decreased in AS- and in CsA-treated cells. Note that in both cases, the decrease is greater for NF-ATc than NF-ATp. Binding was competed by 100x self probe (S) but not a mutant probe (M).

View larger version (30K): [in a new window]   Fig. 6. GATA5 and NF-ATc collaborate in endocardial differentiation. (A) Blocking NF-ATc activation using cyclosporine A (CsA) inhibits TC13 differentiation and reduces GATA5 induction in response to retinoic acid (RA). Immunocytochemistry using the GATA5 antibody was carried out as described in Materials and Methods. Note how cells treated with CsA do not elongate or align (arrowheads) and show reduced but persistant staining for GATA5 (red nuclei) suggesting that GATA5 induction precedes NF-ATc activation in endocardial differentiation. (B) GATA5 and NF-ATc synergistically activate the endothelin 1 (ET-1) promoter. (Top) A schematic representation of the ET-1 promoter showing the close proximity of the NF-ATc and GATA binding sites. (Bottom) A 1.4 kbp endothelial-specific ET-1 promoter is transcriptionally activated by GATA5 and NF-ATc as evidenced in cotransfection with increasing amounts (0.25, 50, 100, 250, 500 ng) of either GATA5 or NF-ATc expression vectors in TC13 cells (left panel). At limiting amount of expression vectors (25 ng) GATA5 and NF-ATc synergistically activate the ET-1 promoter (right panel). The empty pCDNA3 and pCD-SR backbone vectors of GATA5 and NF-ATc, respectively, are used as controls. The results are expressed as fold activation and represent the mean of three independent experiments, each done in duplicate.

View larger version (20K): [in a new window]   Fig. 7. Hierarchy of transcription factors during in vitro endocardial differentiation. Only transcription factors present at the different stages are indicated. Note that GATA5 is the earliest factor that is transcriptionally induced followed by Msx1 and EPAS1. Expression of GATA4 and Tbx20 is maintained, though at a reduced level. Reduced expression of GATA5 following cyclosporine A treatment suggests that calcium signaling may be an upstream GATA5 activator in endocardial cells. The results obtained suggest that GATA5 is required at an early stage of endocardial differentiation. Consistent with in vivo data, NF-ATc transcripts are present from the earliest stages of endocardial commitment, but NF-ATc is activated and required at later stages that remain undefined (Ranger et al., 1998). The role of Msx1 and EPAS1 in endocardial differentiation is still undefined; Msx1 is present in endocardial cushion cells but not in the endocardium, consistent with a role in epithelial-mesenchymal transformation (Chan-Thomas et al., 1993). EPAS-1 is required at terminal stages of endothelial maturation and remodeling (Peng et al., 2000). The appearance of both factors after GATA5 during in vitro endocardial differentiation is consistent with their in vivo distribution and function, at later stages of endocardial differentiation.