Hematopoietic Stem Cells and Multipotent Progenit c-Kit Receptor: Indication of an Alternative c-Kit Signaling Pathway.

The SCF receptor c-kit plays an important role in the maintenance and differentiation of HSC and multipotent progenitors (MPPs). A new truncated, intracellular form of c-kit receptor, called truncated c-kit (tr-kit), was found first in murine male germ cells. Tr-kit is encoded by a 3.2 kb alternative transcript, which originates in the intron 16 of c-kit gene, and contains a unique 415 bp long 5′ UTR and 36 bp long start of the coding sequence. Besides the extracellular, trans-and juxta-membrane region, the 202 aa long tr-kit protein (Mw. 30 kDa) lacks the ATP binding part of the kinase domain, as well as the hydrophilic kinase insert region. Instead, tr-kit contains a unique 12 aa long hydrophobic region, which is in frame with the 190 aa long C-terminal part of c-kit protein, containing the phosphotransferase domain and C-terminal tail, relevant for interaction with PLCγ. Recently, we have detected tr-kit transcript and protein in murine HSC-like cell line EML, which can differentiate into erythroid, myeloid and lymphoid lineages. Moreover, Western analysis with α-c-kit [pY⁹³⁶] phosphospecific Ab has shown that in EML cells tr-kit is phosphorylated at the C-terminal tyrosine Y936, important for interaction with PLCγ. Thus, we have examined tr-kit expression in FACS-purified murine bone marrow (BM) cell populations highly enriched for long-term and short-term repopulating HSC, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, the tr-kit transcript was detected only in the Lin⁻Sca-1⁺c-kit⁺ Flk2⁻, Lin⁻Sca-1⁺c-kit⁺ Flk2⁺ and Lin⁻Sca-1⁺c-kit⁺ BM cell populations, highly enriched for LTR-HSC, STR-HSC and MPPs. On the other hand, the tr-kit transcript was absent in

• more heterogeneous Lin⁻ c-kit⁺ Sca-1⁻, Lin⁻Sca-1⁺ and Lin⁻ Sca-1⁻ BM cells, in which HSC are either present at a low frequency or are absent altogether, and

• Lin⁺ BM cells, DN thymocytes, CD4 and CD8 T cells, pro-B and pre-B cells, monocytes, macrophages and erythroblasts.

To analyze tr-kit expression during differentiation of MPPs into myelo-erythroid lineages, EML cells were cultured in the presence of Epo, GM-CSF, and G-CSF for 72 and 96 hours. The levels of tr-kit transcript and protein were analyzed by quantitative real-time RT-PCR and Western, and the myelo-erythroid differentiation of EML cells was monitored by expression of β-globin and lactoferrin. These experiments have shown that tr-kit transcription and protein expression are quickly down-regulated during myelo-erythroid differentiation of EML cells. More importantly, increased levels of tr-kit protein are associated with SCF-independent maintenance and attenuated differentiation of EML cells, and SCF-independent activation of the full-length c-kit receptor. Together with preferential expression of tr-kit in HSC and MPPs, which also express c-kit, these data suggest that HSC and MPPs could be utilizing distinct SCF-dependent and SCF-independent c-kit signaling. In a proposed alternative model of c-kit function, the maintenance of HSC and MPPs could be mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. Using newly generated tr-kit-specific Ab we are studying interactions of tr-kit with c-kit, PLCγ, and other components of c-kit signaling pathway, and are also analyzing the impact of tr-kit knockdown and over-expression on maintenance and differentiation of EML cells and primary HSC.