1)    Identifying the molecular mechanisms and epigenetic machinery which regulate HSC function and lymphoid lineage specification.
2)    Determining the signaling pathways of chemokine receptors.

CXCR4 in hematopoietic stem cell function:

In order to maintain life-long blood supply, hematopoietic stem cells (HSCs) have to be able to self-renew and differentiate throughout our life. HSCs are located in a specialized microenvironment in the bone marrow, and it is believed that cells in this stem-cell niche produce molecules to control the stem cell functions. However, what the niche cells are, and what molecules they provide to maintain the self-renewal and differentiation capacity of HSCs, remain to be identified. We have found that the chemokine CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor controlling the quiescence of primitive hematopoietic cells. CXCL12, when added into the culture, directly inhibits proliferation of primitive hematopoietic cells. Inactivation of its receptor CXCR4 leads to excessive proliferation of primitive hematopoietic cells. These hyperproliferative CXCR4-deficient primitive hematopoietic cells bear surface makers characteristic for long-term HSCs, are retained in the BM, and sustain hematopoiesis for at least 8 months. More surprisingly, they out-compete the wildtype competitive HSCs in a competitive repopulation assay. Together, these results demonstrate an inhibitory role of the CXCL12-CXCR4 axis in restraining proliferation of HSCs enforced by the CXCL12-expressing cells in the BM niche.

Our results show that inactivation of CXCR4 leads to reduction of expression of the cell cycle inhibitor p57KIP2 in HSCs. Our data suggest a potential signaling circuit for regulating HSC quiescence that connects the signal from BM niche (CXCL12) with a HSC surface receptor (CXCR4) and a cell cycle regulator (p57KIP2). Current projects include investigation the role of p57KIP2 in HSC quiescence using p57KIP2 knockout mice. Using various mouse models, we also aim to understand the relationship of stem cells and their physical niches, and the molecules that regulate the homeostasis of stem cells. Findings will be extended to other stem cells and cancer stem cells.
[back to the top]

Chemokine receptor signaling:

We have been interested in determination of the cellular responses of individual signaling pathways downstream of chemokine receptors. Like all other G-protein coupled receptors (GPCRs), CXCR4 employs two major domains to interact with downstream signaling mediators. One is the highly conserved aspartate-arginine-tyrosine sequence (or the DRY box) located in the second cytoplasmic loop of CXCR4 that is required for coupling to the heterotrimerix G proteins. The other signaling domain of CXCR4 is its serine-rich cytoplasmic tail which has been shown to play an important role in desensitizing the chemokine signaling by internalization of ligand activated receptor.

In order to evaluate the precise function of the two signaling modules of CXCR4, and to ensure physiological expression of the mutant CXCR4, we generated mice in which the endogenous CXCR4 locus was mutated by “knock-in” gene targeting. One mutant strain carried a CXCR4 gene harboring a DRY box replacement mutation (DRYmu), and the other a cytoplasmic tail truncation (∆T). We found that mice expressing ∆T-CXCR4 exhibited phenotypes identical to that of CXCR4-null mice. Our studies further showed that in the absence of the cytoplasmic tail, heterotrimeric G proteins cannot be activated and downstream biochemical events were abolished. These data reveal a critical function of the cytoplasmic tail of CXCR4 in chemokine receptor signaling.

Our recent studies on FAK, a signaling component downstream of chemokine receptors and integrins, have yielded unexpected results. Our data indicate that FAK participates in TCR signaling, as we have found that it is tyrosine-phosphorylated, activated and interacts with LCK following TCR stimulation. T cell development is apparently normal in the FAK-mutant thymus. However, peripheral mature T cells are hypersensitive to TCR stimulation. T cells deficient in FAK are not able to fully differentiate into effector cells upon activation. Instead they rapidly upregulate Fas and undergo apoptosis. Our biochemical data show that FAK prevents CSK protein degradation and thus negatively regulates LCK activity. On the other hand, FAK enhances PLCγ activity and subsequently controls Ca2+-dependent signaling and the NFκB pathway. Thus, FAK coordinates divergent signaling pathways to tune TCR signaling threshold, allowing for efficient generation of effector T cells.
[back to the top]