Gal-3 induces the transcription of CCL2 and BSG through NFAT1 dephosphorylation. (IMAGE)
Caption
(A) Cytosolic calcium dynamics in HPSCs were monitored via treatment with the calcium dye Fluo-4/AM with or without Gal-3 (50 ng/mL). Gal-3 treatment significantly increased the fluorescence intensity. (B) Quantification of calcium intensity in random frame phases revealed the function of Gal-3, shown in Figure 3A. (C) Time-lapse imaging of individual cells revealed greater peaks and more frequent calcium oscillations in Gal-3-treated cells than in control cells, indicating a distinct transient calcium phenotype induced by Gal-3. (D) Quantification of the number of calcium oscillations per minute in response to Figure 3C. (E) The expression of PPP3CA (calcineurin A) was significantly increased in the cytoplasm of Gal-3-treated cells, as shown by imaging flow cytometry. (F) CALN activity was similarly increased in Gal-3 genetic HPSCs, confirming enhanced calcium signaling. (G) Imaging flow cytometry revealed that Gal-3 treatment induced the translocation of NFAT1 from the cytoplasm to the nucleus. (I) JASPAR database enrichment of the potential NFATC2 consensus motif (TTTTCCA) at the promoters of CCL2 and BSG. (J) Luciferase assays revealed that NFATC2 activated the transcription of CCL2 and BSG in a dose-dependent manner following co-transfection with the pGL3-CCL2-luc and pGL3-BSG-luc vectors in HEK293T cells. (K) Chromatin immunoprecipitation‒PCR results demonstrated that NFATC2 bound to the promoter regions of CCL2 and BSG in HPSCs. Two primers for BSG were designed to separate the two bands at the promoter of NFATC2. (L) The dual-luciferase reporter plasmids CCL2 and BSG were co-expressed with NFATC2 in HEK293T cells. (M) The efficiency of siRNA-mediated knockdown of NFATC2 in reducing the RNA levels of CCL2 and BSG. (N) siNFATC2 significantly reduced CCL2 secretion, as shown by ELISA. (O) NFATC2 knockdown decreased the accumulation of BSG on the cell membrane, as determined by a flow cytometry assay. (B), (D), (F), (J), and (L–N) represent the mean ± standard deviation of independent experiments. p-values were analyzed via two-tailed unpaired student's t-tests for (B), (D), (F), and (N) and one-way ANOVA for (J), (L), and (M). “∗" indicates statistical significance. Gal-3, galectin-3; CCL2, C–C motif chemokine 2; PPP3CA, protein phosphatase 3 catalytic subunit alpha; NFATC2, nuclear factor of activated T-cells 2; BSG, basigin; NFAT1, nuclear factor of activated T-cells 1; HPSCs, human pancreatic stellate cells; CALN, calcineurin.
Credit
Yaheng Wu, Guo An, Jia Tong, Wenlong Zhang, Zhihua Tian, Bin Dong, Xijuan Liu, Lin Zhao, Chunxian Ye, Jingtao Liu, Wei Zhao, Huachong Ma
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