Mechanisms associated with IL-6–induced up-regulation of Jak3 and its role in monocytic differentiation

Abstract

We report here that Janus kinase 3 (Jak3) is a primary response gene for interleukin-6 (IL-6) in macrophage differentiation, and ectopic overexpression of Jak3 accelerates monocytic differentiation of normal mouse bone marrow cells stimulated with cytokines. Furthermore, we show that incubation of normal mouse bone marrow cells with a JAK3-specific inhibitor results in profound inhibition of myeloid colony formation in response to granulocytemacrophage colony-stimulating factor or the combination of stem cell factor, IL-3, and IL-6. In addition, mutagenesis of the Jak3 promoter has revealed that Sp1 binding sites within a ؊67 to ؊85 element and a signal transducer and activator of transcription (Stat) binding site at position ؊44 to ؊53 are critical for activation of Jak3 transcription in murine M1 myeloid leukemia cells stimulated with IL-6. Electrophoretic mobility shift assay (EMSA) analysis has demonstrated that Sp1 can bind to the ؊67 to ؊85 element and Stat3 IntroductionThe development of hematopoietic precursor cells into terminally differentiated cells such as granulocytes and macrophages requires integration of signals for cell survival, growth, and differentiation. These signals are induced by a network of cytokines, which bind to their receptors and initiate signals that can couple the processes of growth and differentiation. 1 Many cytokines transmit their signals through a family of cytoplasmic tyrosine kinases known as Janus kinases (Jak kinases). 2 Gene targeting studies in mice have illustrated the importance of the Jak family members in hematopoiesis. Lymphoid development is impaired in mice deficient in Jak1 3 and Jak3, [4][5][6] and erythropoiesis is lost in Jak2-deficient mice. 7 Among the Jak family members, Jak3 is unique because it is expressed mostly in hematopoietic cells and its transcription is regulated by cytokines such as granulocyte colony-stimulating factor 8 (G-CSF), which can induce terminal differentiation of myeloid cells. In addition, Jak3 transcription was shown to be up-regulated by interleukin-2 (IL-2) and IL-4, which mediate proliferative signals in T and B cells. [9][10][11][12][13] Interestingly, overexpression of Jak3 in either 32Dcl3 cells or primary mouse bone marrow (BM) cells in the presence of G-CSF or granulocyte-macrophage (GM)-CSF results in an early onset of growth arrest of the cells in the G 1 phase of the cell cycle, which is accompanied by terminal granulocytic differentiation. 8 It thus appears that Jak3 can transmit either proliferative or growth arrest signals depending on the cell type and the nature of the cytokine used. In this study, we present evidence that Jak3 also plays an important role in myeloid differentiation along the monocytic lineage. Furthermore, detailed analysis of the promoter/enhancer region of Jak3 has revealed that regulation of Jak3 transcription during IL-6-induced macrophage differentiation is achieved through a mechanism that is distinct from the mechanism of activation of the JAK3 promoter (JAK3pr) in activated T cells that ...

Granulocyte colony-stimulating factor-induced upregulation of Jak3 transcription during granulocytic differentiation is mediated by the cooperative action of Sp1 and Stat3

“…Our model reveals the possibility that activation of STATs by cytokines is not an end point, but rather that activated STATs can then bring about the activation of a second Jak kinase pathway with distinct consequences. We also observed similar regulation of Jak3 transcription during IL-6-induced macrophage differentiation (Mangan et al, 2004). This suggests that Sp1 and Stat3-induced activation of Jak3 transcription is a common pathway in both granulocytic and monocytic differentiation.…”

“…Site-specific mutation of these elements, electrophoretic mobility shift assays, and transcriptional transactivation studies demonstrate a critical role for these Sp1 and Stat-binding sites in mediating Jak3 transcription during granulocytic differentiation induced by G-CSF. This is similar to a mechanism we observed during IL-6-induced macrophage differentiation of M1 myeloid leukemia cells (Mangan et al, 2004), but differs from a STAT-independent mechanism of activation of JAK3 transcription that occurs in activated human T cells (Aringer et al, 2003).…”

“…To gain an insight into the molecular mechanisms associated with Jak3 transcription in response to G-CSF, we created several 5 0 deletion mutants of the Jak3 promoter and subcloned these deletion mutants, the longest of which contained 3.8 kb upstream of the transcriptional start site, into the pGL3-Basic luciferase reporter vector. These constructs have been described previously (Mangan et al, 2004). The deletion mutants that were used in the luciferase reporter assays are depicted in Figure 1.…”

“…The coding region of Jak3 was replaced with the coding region of the firefly luciferase gene by subcloning a 5.7 kB genomic clone and several 5 0 deletion constructs into the pGL3-Basic luciferase reporter vector, and these deletion constructs are depicted here. The constructs have been designated Jak3 promoter À3805, Jak3 promoter À2296, etc to indicate the position of the 5 0 end of the deletion mutant relative to the start site of transcription, as described previously (Mangan et al, 2004). The pGL3-Basic Jak3pr À3805 construct and several 5 0 deletion constructs were transiently transfected into 32Dcl3 cells that were either left unstimulated or were stimulated with G-CSF.…”

“…The promoter/enhancer sequences upstream of the ATG codon were then cloned into the pGL3-Basic promoterless vector upstream of the luciferase reporter gene (Promega, Madison, WI, USA). This construct has been described previously (Mangan et al, 2004).…”