Cytokine signals through STAT3 promote expression of granulocyte secondary granule proteins in 32D cells

Abstract

Objective: In a previous study, we showed that activation of a transfected human erythropoietin receptor (EPOR) in the murine myeloid cell line 32D resulted in the development of morphologic features of granulocytic differentiation and expression of the neutrophil primary granule protein myeloperoxidase. We now studied if EPOR signaling could also mediate secondary granule protein gene expression and investigated the signal transduction requirements for induction of secondary granule gene expression in 32D cells.

Materials and methods: Wild-type and variant 32D cells expressing normal or chimeric EPORs or receptors for granulocyte colony-stimulating factor (G-CSFRs) were stimulated with EPO or G-CSF and the expression of granulocyte-specific genes was analyzed by Northern blot analysis. To determine the signaling mechanisms required for secondary granule protein gene induction, the activation of STAT pathways following growth factor stimulation was studied by Western blot analysis.

Results: We found that EPO treatment of 32D cells engineered to express EPOR did not result in induction of the secondary granule protein genes encoding lactoferrin and 24p3 lipocalin, the mouse homolog of human N-Gal, or the myeloid transcription factor C/EBPepsilon. Replacement of the intracellular domain of EPOR with the intracellular domain of G-CSFR in a chimeric receptor was associated with EPO-mediated induction of lactoferrin, 24p3 lipocalin, and C/EBPepsilon genes. We found that STAT3 phosphorylation was mediated by the intracellular domain of G-CSFR, but not EPOR. Replacement of one or two of the STAT5 binding sites in the intracytoplasmic domain of the EPOR with STAT3 binding sites resulted in EPO-mediated STAT3 activation and a marked increase in the expression of the 24p3 lipocalin gene. Knockdown of STAT3 protein levels with siRNA caused significant decrease in 24p3 lipocalin gene induction.

Conclusion: These results indicate that EPOR signaling cannot substitute for G-CSFR signaling to stimulate secondary granule protein gene expression in 32D cells. In addition, STAT3 is a critical mediator of 24p3 lipocalin gene expression in these cells.

Figure 1

Expression of the mRNAs encoding the primary granule protein myeloperoxidase and secondary granule proteins lactoferrin and 24p3 lipocalin in 32D-derived cell lines after EPO stimulation. The cells were cultured in medium containing 2 units/mL of EPO for the indicated times. Ten ug of total cellular RNA was analyzed by Northern blot hybridization, using as probes ³²P-labeled murine cDNAs encoding MPO (A), L F or 24p3 lipocalin (B), and β-actin as a control for the amount of sample. FE: 32DEPOR-FE cells, expressing a C-terminal truncated human EPOR; WT: 32DEPOR-WT cells, expressing wild-type human EPOR; wt18: 32Dwt18 cells, expressing a chimeric EPOR/G-CSFR (see Materials and methods).

Figure 2

Expression of C/EBPα and C/EBPε mRNA in 32D-derived cell lines after EPO stimulation. Cells were cultured in medium containing 2 units/mL of EPO for the indicated times. Northern blot analysis was carried out as in Figure 1, using as probes ³²P-labeled murine cDNAs encoding C/EBPα, C/EBPε, or β-actin. Cell line designations as in Figure 1.

Figure 3

Induced phosphorylation of Stat3 and Stat5 in 32D-derived cell lines after EPO stimulation. Cells were cultured in medium containing 10 units/mL of EPO for the indicated times. Whole-cell lysates were fractionated by SDS-PAGE and analyzed by Western blotting using anti-phospho-Stat3 or anti-phospho-Stat5 antibody. The membranes were then stripped and reprobed with anti-STAT3 or anti-STAT5 antibody. Cell line designations as in Figure 1.

Figure 4

Schematic diagram of the structure of the murine EPOR expressed in cell lines WT EPOR, ER343-S3, and ER343/401-S3. The cytoplasmic region of the murine wild-type (WT) EPOR is diagrammed on the left, showing the position of the JAK2 binding domain (cross-hatched bar) and the regions surrounding the tyrosine residues required for STAT5 binding and activation (Tyr-343 and Tyr-401). ER343-S3 has the sequence GYMPQ (STAT3 binding site) substituted for residues 342–346 of the WT murine EPOR. ER343/401-S3 has the STAT3 binding site substituted at residues 342–346 and 400–404 of the WT murine EPOR. Adapted from Wooten et al. [35].

Figure 5

Pattern of Stat3, Stat5, and JAK2 phosphorylation in 32D-derived cells after EPO stimulation. Western blot analysis was carried out as described in Figure 3. Cell line designations as in Figure 1 (wt18) and Figure 4 (WT EPOR and ER343-S3).

Figure 6

Gene expression of secondary granule protein 24p3 lipocalin in 32D-derived cell lines after EPO stimulation. Northern blot analysis was carried out as described in Figure 1. Cell line designations as in Figure 4.

Figure 7

Expression levels of STAT3 protein and 24p3 lipocalin mRNA in EPO-stimulated ER343-S3 and 32Dwt18 cells after transfection with siRNA. Cells were transfected with siRNA by electroporation, cultured in medium containing IL-3 for 5 hours, then washed and transferred to medium containing 2 units/mL of EPO and cultured for an additional 19 hours. Western blot analysis (A) and Northern blot analysis (B) were carried out as described in Figure 3 and Figure 1, respectively. Quantitation of STAT3 protein and 24p3 lipocalin mRNA was performed by PhosphorImager using the ImageQuant software program (Molecular Dynamics) and the average values of the siRNA/control (CT) ratios from at least two separate experiments are shown below the corresponding STAT3 and 24p3 lipocalin images. Cell line designations as in Figure 1 (wt18) and Figure 4 (ER343-S3).

Figure 8

Phosphorylation of STAT3 and STAT5 and expression of 24p3 lipocalin mRNA in 32D-derived cell lines after EPO stimulation. Western blot analysis (A) was carried out as described in Figure 3 and Northern blot analysis (B) was carried out as described in Figure 1. Cell line designations as in Figure 4.

Figure 9

EPO-induced 24p3 lipocalin mRNA expression in ER343/401-S3 cells in the presence or absence of the protein synthesis inhibitor cyclo-heximide (CHX). ER343/401-S3 cells (Fig. 4) were cultured medium containing IL-3, then washed and transferred to medium containing 5 μg/L of CHX, 2 units/mL of EPO, or both EPO + CHX. Cells were harvested at the indicated times, and their total RNA extracted for Northern blot analysis, carried out as described in Figure 1.

Figure 10

Expression of 24p3 lipocalin mRNA in EPOR WT and ER343-S3 cells after stimulation by EPO in the presence or absence of 10 μM ATRA. EPO stimulation and Northern blot analysis were carried out as described in Figure 9. Cell line designations as in Figure 4.