In an attempt to understand the biological roles of RTK-mediated Src family kinase and PI 3-kinase signaling in vivo we have characterized the phenotypes of mice expressing mutant Kit receptors, which fail to activate PI 3-kinase or Src family kinase signaling, KitY719F
, obtained by a knock-in strategy. Blockade of either of the two signaling pathways produces phenotypes in distinct cell populations in early B and T cell development, in mast cells, and in spermatogenesis. But in most other cell types, mutant phenotypes are minor. Similar mutational analysis of PDGF receptor α chain signaling in vivo in which the analogous PI 3-kinase and Src binding sites were mutated gave rather different results (30
). On one hand the PI 3-kinase binding site mutation produced severe phenotypes in many cell types. In contrast, the Src binding site mutation had a limited phenotype in oligodendrocyte development. These results would suggest that PDGF-mediated PI 3-kinase signaling is critical for PDGF function in vivo. Therefore, analogous mutations in the closely related Kit and PDGF receptor α chain have very different in vivo consequences. This raises the possibility that the cellular context in which the receptor functions may have a very critical role.
Whereas the Y719F mutation blocks the direct binding by Kit of the p85 regulatory subunit of PI 3-kinase and its activation, the Y567F mutation blocks Src kinase family binding and activation as well as binding of the tyrosin phosphatase SHP2 and APS family adaptor proteins (14
). Therefore, the interpretation of the consequences of the Y567F mutation is quite complex as three different signaling events have to be considered as well as the specific cellular context. The Src family of kinases includes Src, Yes, Lyn, Fyn, Blk, Lck, and Fgr. Whereas, Src and Yes are expressed ubiquitously, the other members are expressed in a tissue-specific manner and they may activate distinct cellular responses (31
). Therefore, the Y567F mutation may block activating as well as inhibitory signaling events related to cell proliferation, cell survival as well as receptor desensitization.
Both the KitY567F
and the KitY719F
mutation affect Kit function only in specific developmental processes and this is in contrast to other mutations in the Kit receptor gene that broadly affect Kit function in hematopoiesis, gametogenesis, and melanogenesis. Thus, both mutations fail to affect steady-state hematopoiesis and tissue mast cell numbers, but they substantially reduce peritoneal mast cell numbers. Furthermore, although Kit has important functions at multiple stages in embryonic and postnatal gametogenesis, the KitY719F
mutation, and thus PI-3 kinase signaling is critical only in a specific subset of the postnatal stages in the ovary and testis, but the KitY567F
mutation does not affect these processes. The KitY719F
but not the KitY567F
mutation affects activation of the PI 3-kinase Akt signaling cascade known to have a critical role in mediating cell survival (unpublished data), thus during the spermatogonial stages Kit signaling appears to be critical in mediating cell survival (18
). We conclude, therefore, that in most cells that require Kit, there are redundant signaling pathways, but that in certain cell types, the PI-3 kinase pathway is critical.
In embryogenesis and in neonatal mice T cell development is reduced, but permissive, and B cell development is unaffected by loss of Kit function. In adult mice, recent studies demonstrated a role for Kit function in both B and T cell development (9
). In viable Kit-null mutant mice an age-dependent progressive decline of pro B and pro T cells was observed with a concurrent loss of common lymphoid progenitors in the bone marrow.
In B cell development, the Kit-null mutation, KitW
, does not affect the immunoglobulin repertoire and the establishment of the peripheral B cell compartment, but Kit function is required for the maintenance of B lymphopoiesis from hematopoietic stem cells in adult mice (9
). In KitY567F/Y567F
, but not in KitY719F/Y719F
mice, we demonstrate a partial block during the Kit-positive pro B cell stages in agreement with these earlier results. Whereas, hematopoietic stem cells and early progenitor numbers in the BM determined by FACS®
are normal in both KitY567F/Y567F
and in KitY719F/Y719F
mice (unpublished data), in BM of KitY567F/Y567F
mice there was a progenitor deficit with reduction in fraction C and D, but mature recirculating B lymphocytes were not significantly reduced. It is of interest to note that this phenotype emerges late in life, i.e., it is detectable at 9 mo and becomes more prominent in older mice. Young adult animals (4-mo-old) are not affected. It is possible that an age-related change in the bone marrow microenvironment may contribute to the expression of this phenotype. Thus, signaling mediated by phosphorylation of KitY567
is important for Kit function in pro B cell development in an age-dependent fashion. Identity of the possible Src family kinase members involved in this is not known. In addition, contributory roles of SHP-2 and the APS adaptor are difficult to evaluate as well. However, Kit-mediated activation of PI 3-kinase does not appear to be critical. In contrast to spermatogenesis in lymphocyte development a Kit-mediated survival signal is either not critical or compensated for by another signaling mechanism. The signal provided by Kit-PY567, which is critical for lymphocyte development, on the other hand may effect either cell proliferation and/or differentiation.
In T cell development loss of Kit function over time produces a progressive loss of pro-thymocytes. There is an age-dependent block at the TN2 stage and consequently accumulation of CD44+
TN1 cells as well as a reduction of double-positive and single-positive thymocytes (9
). In thymi of KitY567F/Y567F
, but not KitY719F/Y719F
mice, analysis of lineage-negative thymocytes showed an increase of TN1 cells and a corresponding decrease of the CD25+
TN3 cell populations. But similar to thymi of mice carrying the hypomorphic Kit allele KitWv
, single- and double-positive thymocyte numbers in KitY567F/Y567F
thymi were not affected. In T cell development age also plays an important role. A mutant phenotype is first detected at 6 mo and is more severe at 9 mo. Thus, the effect of the KitY567F
mutation on thymocyte development is partial and age related. In contrast the KitY719F
mutation does not affect thymopoiesis.
Mast cells arise from progenitors in the bone marrow, however, maturation and differentiation of these cells occurs mainly in tissues where they reside (33
). Kit has a major role in mast cell development and mast cell function and mice with Kit loss of function mutations lack mast cells in various tissues of the adult organism including skin, mucosa of the GI tract, lung, peritoneal cavity, and its associated mesentery. Interestingly, in both KitY719F/Y719F
mice mast cell numbers in dorsal skin sections were not reduced. In contrast, both mutations affected peritoneal mast cell numbers to differing degrees. Importantly, remaining peritoneal mast cells in the two mutant mice were fully differentiated. Quite likely then the peritoneal microenvironment may not be able to compensate the Kit signaling deficiencies.
The tyrosine kinase inhibitor STI571/imatinib is a specific inhibitor for the ABL, ARG, PDGFR, and Kit kinases. It is used to treat chronic myelogenous leukemia and gastrointestinal stromal tumor patients with considerable success. An important clinical attribute of this drug is the lack of major known side effects. Since imatinib also inhibits kinases other than Kit, notably ABL, blocking their function may also contribute to the phenotypes in mice treated with imatinib. A role for ABL in B cell development has been inferred from phenotypes of mice with an ABL-null mutation (35
). However, we consider this possibility unlikely because (a) the phenotypes in pro B cell development in ABL mutant mice are quite variable and affect all pro B cell subsets, and (b), perhaps more importantly, treatment of mice with imatinib produces similar, age-dependent pro T and pro B cell phenotypes as those seen in mice carrying germline Kit mutations (this paper and reference 9
). The overlapping phenotypes caused by genetic and pharmacological ablation suggest that both genetic and pharmacological modes of Kit inhibition affected the same signaling pathways in vivo. Moreover, similar to mice carrying germline Kit mutations, which lack Kit function throughout live, our data using Imatinib show that acute inhibition of Kit signaling in older mice rapidly affects lymphopoiesis. The increased Kit dependency of lymphopoiesis with age may result from age-dependent changes in the bone marrow and the thymic microenvironments such that Kit signaling becomes more critical. The molecular nature of the age-associated changes remains to be determined. The effect of STI571 on the immune system's response to an antigen has been assessed in a 4-week study in rats (8-week-old). STI571 was administered orally up to 60 mg/kg/d. Relative counts of lymphocyte subpopulations in peripheral blood, thymus, spleen, and mesenteric lymph nodes revealed no obvious changes attributable to either administration of STI571 or immunization of animals with KLH. There were no findings indicative of an adverse immunotoxicological effect (Paul, G.R., U. Junker, and P. Ulrich, personal communication).