Molecular mechanisms associated with upregulation of Jak3 transcription during myeloid differentiation

We previously demonstrated that Jak3 is a primary response gene for G-CSF during granulocytic differentiation and a primary response gene for IL-6 during monocytic differentiation. To understand the molecular mechanism underlying the regulation of Jak3 transcription induced by G-CSF or IL-6, we performed luciferase reporter assays in the murine myeloid cell line 32Dcl3, with or G-CSF stimulation, and the murine myeloid leukemia cell line M1, with or without IL-6 stimulation. In G-CSF-stimulated 32Dcl3 cells and IL-6-stimulated M1 cells, mutation of either a -67 to -85 element or a -44 to -53 GAS element resulted in a marked reduction of Jak3 promoter activity. Electrophoretic mobility shift assays (EMSAs) revealed that Spl present in nuclear lysates of both 32Dc13 cells stimulated with G-CSF and M1 cells stimulated with IL-6 can bind to the -67 to -85 element. Likewise, EMSA analysis using nuclear lysates from G-CSF-stimulated 32Dc13 cells or IL-6-stimulated M1 cells demonstrated that STAT3 can bind to the -44 to -53 GAS element. Co-transfection of a constitutively active mutant of STAT3 into G-CSF-stimulated 32Dcl3 cells or IL-6-stimulated M1 cells along with a Jak3 promoter/luciferase reporter resulted in enhancement of Jak3 promoter activity. Together, these results demonstrate that activation of Jak3 transcription during G-CSF-induced granulocytic differentiation or IL-6-induced monocytic differentiation is mediated by the combined action of Sp1 and STAT3.; In addition we observed strong Jak3 promoter activity in 32Dcl3 cells proliferating in the presence of IL-3. The strong Jak3 promoter activity in conditions of IL-3-stimulation was surprising, since Jak3 mRNA is poorly induced by IL-3. It is likely that an as yet unidentified post-transcriptional mechanism accounts for the weak induction of Jak3 mRNA in IL-3-stimulated 32Dc1l3 cells despite strong activation of the Jak3 promoter in these conditions. During myeloid differentiation, however, G-CSF or IL-6 strongly induces activation of Jak3 transcription, which is mediated by the cooperative action of Sp1 and STAT3. This is distinct from the mechanism recently reported to upregulate Jak3 transcription in activated T cells.