ALBERT

Description: It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune conditions suggest that the CD6–CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.

Description: Intrinsic mechanisms that regulate self-renewal of mammalian stem cells remain largely unknown. Stem cell maintenance and self-renewal in Drosophila and C. elegans are regulated by members of the conserved Pumilio family of RNA-binding proteins. We have previously described cloning and characterization of two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the role of mammalian Pum proteins in HSC, Pum2 was over-expressed in a SCF-dependent multipotent progenitor cell line EML, which has the capacity for multilineage (erythroid, myeloid, B and T lymphoid) differentiation in vitro. In the presence of SCF EML cells undergo SCF-dependent self-renewal, thus remaining undifferentiated and retaining an immature phenotype. When cultured with hematopoietic cytokines (IL-3, GM-CSF, Epo, Tpo) EML cells differentiate into lineage-committed hematopoietic progenitors (e.g. granulocyte/macrophage (CFU-GM), burst-forming unit erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors). Pum2 over-expression leads to uncoupling of the survival and differentiation signals in EML cells, and their SCF-independent maintenance. EML cells over-expressing Pum2 (Pum2-EML cells) also exhibit almost complete block of differentiation into multiple lineages in the absence of SCF. Moreover, although the culture with cytokine cocktail (IL-3, Epo, Tpo and GM-CSF) and retinoic acid enhances differentiation capacity of wild type EML cells, it was not sufficient to overcome the differentiation block in Pum2-EML cells. However, the repression of Pum2-EML cell differentiation is a reversible phenomenon, since the addition of SCF to Pum2-EML cell cultures, for at least 48 hours, restores their capacity to undergo multilineage differentiation and generate hematopoietic colonies. The SCF-independent maintenance of Pum2-EML cells seems to be caused by upregulated expression and constitutive activation of the SCF receptor c-kit, and is accompanied by constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. More importantly, Pum2-EML cells also exhibit upregulated expression and constitutive activation of a novel truncated form of c-kit receptor called tr-kit, which was found previously to be expressed preferentially in HSC. Tr-kit could play a critical role in the SCF-independent activation of the full-length c-kit receptor, leading to SCF-independent maintenance of Pum2-EML cells, and inhibition of their multilineage differentiation. The observation that Pum2-EML cells, maintained with or without SCF, are resistant to treatment with blocking anti-c-kit antibody (ACK2) and c-kit inhibitor STI-571, supports the notion that maintenance and survival of Pum2-EML cells in the presence of SCF is not due to an external activation of c-kit receptor through ligand binding. Taken together, these findings suggest a model in which survival and maintenance of multipotent hematopoietic progenitors are mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. In summary, mouse Pum2 protein could play an important role in supporting maintenance of HSC and multipotent progenitors through regulation of the SCF/c-kit signaling pathway, and could represent a part of the mechanism through which HSCs balance their self-renewal and commitment to differentiation.

Description: 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 inmore 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, andLin+ 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.