In this study we have found that FL preferentially activates Stat5a over Stat5b, and that myeloid progenitors from the bone marrow of Stat5a+/+, Stat5b+/+, and Stat5b−/−, but not from Stat5a−/−, mice respond to the stimulating/costimulating effects of FL. These results demonstrate that Stat5a plays a critical role in mediating FL effects. Moreover, this adds additional evidence to the literature showing the nonoverlapping effects of Stat5a and Stat5b.
Among the different Stat proteins examined, only Stat5a was potently tyrosine phosphorylated by FL stimulation. FL did not seem to activate any Janus kinase as measured by phosphotyrosine immunoblotting (). Longer stimulation with FL (30 min) did not result in any phosphorylation of Jak1 and Jak2 (data not shown). This is in contrast to most growth factors whose receptors are tyrosine kinases, such as PDGF, SLF, and M-CSF (or CSF-1). PDGF, SLF, and M-CSF have been shown to activate multiple Jak and Stat proteins. M-CSF induces tyrosine phosphorylation of Jak1, Tyk2, and Stat1, 3, and 5; PDGF activates Jak1, Jak2, Jak3, and Stat1, 3, 5, and 6 41424344
; and SLF activates Jak2 and Stat1, 3, and 5 4546
. These growth factors activate both Stat5a and Stat5b. Our results suggest that FL differs from these RTKs in that it activates only Stat5a without an apparent activation of Jaks.
Recently, a group reported that a tandem-duplicated form of Flt3 constitutively activates Stat5 and mitogen-activated protein (MAP) kinase, whereas FL stimulation leads to activation of MAP kinase but not Stat5 activation in Baf3 and 32D cells expressing wild-type Flt3 47
. In that study 47
, it was not specified whether Hayakawa et al. were dealing with Stat5a or Stat5b or both Stat5 proteins. Our data show that FL stimulation of wild-type Flt3 preferentially activates Stat5a, and our in vivo data strongly suggest that Stat5a, but not Stat5b, plays a critical and potentially physiological role in mediating the synergistic effects of FL. Although the same cell line, Baf3, was used, the discrepancy between the study by Hayakawa et al. and ours may be due to different levels of Flt3 expression and the assays used.
Stat5a and Stat5b are two highly related transcription factors encoded by two different genes. Stat5a and Stat5b are activated by a broad range of cytokines and growth factors. Although most cytokines and growth factors activate both Stat5a and Stat5b, there are some studies showing that Stat5a and Stat5b can be differentially activated and regulate different sets of genes. For example, GM-CSF preferentially activates Stat5b, but not Stat5a, in human neutrophils, whereas IFN-α and -γ predominantly activate Stat5a in promonocytic U937 cells 4849
. Insulin preferentially activates Stat5b, but not Stat5a, via a Jak2-independent signaling pathway in kym-1 rhabdomyosarcoma cells 50
. Not only can Stat5a and Stat5b be differentially activated, they also have distinctive functions. In support of this, Stat5a−/−
mice have different phenotypes 2223242551
. Our findings that FL preferentially activates Stat5a in both Baf3/Flt3 and COS7 cells and that myeloid progenitors from the marrow of Stat5a+/+
, and Stat5b−/−
, but not from Stat5a−/−
, mice respond to the stimulating/costimulating effects of FL provide another example of differential activation of Stat5 protein. Moreover, our results strongly suggest that Stat5a, but not Stat5b, plays a critical role in myeloid progenitor cell responsiveness to the stimulating/costimulating effects of FL.
The Jak/Stat pathway has been well characterized in cytokine signaling. In the type I cytokine receptor family, ligand stimulation induces rapid activation of Jaks which then phosphorylate Stat proteins. In contrast, in tyrosine kinase receptors, the role of Jak proteins in Stat activation is not clearly defined. Some RTKs like PDGFR and epidermal growth factor receptor (EGFR), although they can activate Jaks, activate Stat proteins in a Jak-independent fashion 4352
. In contrast, the activity of the intrinsic RTK is absolutely required for Stat activation by EGF or PDGF. A mutant PDGFR, which lacks kinase activity, is unable to stimulate Stat1 and Stat3 activation in response to PDGF stimulation 43
. Stat1 can directly interact with EGF, PDGF, and SLF receptors and these receptors can phosphorylate Stat1 in vitro 465354
. EGFR can also directly phosphorylate Stat3 in vitro 55
. These results suggest that Stat proteins may be direct substrates of receptor tyrosine kinases. Our findings that the kinase activity of Flt3 is required for Stat5a activation supports this hypothesis. However, our results do not rule out the possibility that Flt3 may activate an endogenous tyrosine kinase that in turn phosphorylates Stat5a. Regardless of the mechanism, our data confirm that Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their functions.