STAT3-activated CD36 facilitates fatty acid uptake in chronic lymphocytic leukemia cells

Figures

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  Figure 1. CLL cells express high levels of CD36 (A) Western blot analysis of CLL cells from 6 different patients and CD19+ normal B cells from 2 normal controls. In all patients samples CD36 was detected at higher levels than in normal B cells. Phosphoserine STAT3 was detected in all patient samples but not in CD19+ normal B cells and levels of unphosphorylated STAT3 were higher in CLL patient samples than in CD19+ normal B cells. Actin was used as loading control and densitometry units were normalized to actin units in the corresponding lanes. (B) CLL cells co-express CD19, CD5 and CD36. Left panel: A representative figure of flow cytometry analysis performed on CLL cells from 6 different patients is depicted. Right panel: Isotype controls. The black line is Isotype control and the red line is the corresponding antibody. Percentages represent the differences between the isotype and the specific antibody. The precentages are the (C) Confocal microscopic images (×400) of freshly isolated CLL cells (right) and normal B cells (left) stained with anti-CD36 antibodies (green) and Evans blue (red) showing CD36 on the cell surface of CLL cells but not on normal B cells.

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  Figure 2. STAT3 binds to and activates the CD36 gene promoter (A) Sequence analysis of the CD36 gene promoter upstream of the start codon (green) revealed 4 putative STAT3 binding sites (red). Upstream putative STAT3 biding sites are not shown because in most experiments we concentrated our efforts on studying binding sites that are in proximity to the CD36 start codon. (B) Using the ChIP method, CLL-cell chromatin fragments pulled down by anti-STAT3 antibodies were analyzed by PCR using primers directed at the 4 putative STAT3 binding sites upstream the CD36 gene start codon. As shown, anti-STAT3 antibodies co-immunoprecipitated the DNA detected by primers +236 bp – +406 bp, +120 bp – +256 bp (amplifying regions of the STAT3 putative binding sites +187 bp – +196 bp, +363 bp – 374 bp, and +382 bp – +391 bp), and −527 bp – −355 bp but not by primers −203 bp – +1 bp (amplifying the region of the putative binding site −93 bp – 83 bp). (C) Using EMSA, biotin-labeled CD36-DNA probes were incubated with CLL cells’ protein extract from 2 patients. The EMSA demonstrated that CLL cell nuclear protein extracts bound to the CD36 gene promoter at regions that include the putative STAT3 binding sites…

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  Figure 3. CLL cell FA intake and metabolism is CD36-dependent (A) CLL cells were transfected with CD36-siRNA or GAPDH and incubated with 80 mM oleic acid in a serum-free, glucose-free medium in tightly sealed flask for 48 hours. Transfection (at a transfection efficiency of 30% as assessed by flow cytometry; left panel) with CD36-siRNA, but not GAPDH, significantly reduces CD36 mRNA levels compared to transfection with GAPD (CTRL), and the dO2 levels were significantly reduced in the medium of non-transfected or GAPDH-transfected CLL cells but not in the medium of CD36-siRNA-transfected cells (right panel). Representative results from 3 different experiments are depicted. (B) CD36 neutralizing antibodies inhibited FA metabolism in CLL cells. CLL cells from two patients were incubated in the presence or absence of 80 mM palmitic acid (PA) or oleic acid (OA) with or without CD36 neutralizing antibodies (Ab) in sealed tissue culture flasks. The relative dO2 in the culture media was assessed prior to and after 48 hours of incubation. All control cultures contained ethanol at the same concentration as in palmitic acid and oleic acid. To compare dO2 we assessed the difference in O2 concentration…