Adhesion Receptors as Regulators of the Hematopoietic Process

HEMATOPOIESIS IS A complex process in which pluripotent stem cells proliferate and differentiate to generate the full complement of mature blood cells. More than 30 hemopoietic cytokines and growth factors that increase or decrease progenitor proliferation and differentiation have been cloned and characterized.1-3 Although the biological effects of these cytokines and growth factors on stem and progenitor cells has been extensively studied, we still do not understand how this extremely orderly hematopoietic process is regulated. Under steady-state conditions, hematopoiesis takes place within the bone marrow microenvironment. Stem cells and their progeny interact relatively specifically with cell and extracellular matrix (ECM) ligands present in the marrow but not other microenvironments.4-6 These adhesive interactions are responsible for the retention of hematopoietic cells in the marrow. Like hematopoietic cells, hematopoietic cytokines and growth factors bind to some specific extracellular matrix components7,8 and stromal cells can express certain cytokines on their surface.9 Selective adhesion of progenitors and cytokines to ECM components or stromal cells then results in the colocalization of progenitors at a specific stage of differentiation with a specific array of cytokines, in so-termed niches.10,11 This provides one level of growth and differentiation regulation. There is also mounting evidence that contact interactions per se between progenitors and marrow stromal ligands play an important role in the regulation of the hematopoietic process.12-14 Adhesive interactions themselves may serve as growth or survival signals or adhesion itself may modulate cytokine- or growth factor-dependent signals. These contact-mediated cues may be responsible for regulating the orderly progression of hematopoiesis.

More than 20 different adhesion receptors have been identified on hematopoietic progenitors.15,16 These include members of the integrin family responsible for adhesion to ECM components (fibronectin, collagen, laminin, or thrombospondin)17 or cell surface-expressed cell adhesion molecules (CAM; vascular [VCAM] and intracellular [ICAM]).18,19 Progenitors express CD44,20 which supports adhesion to hyaluronate20,21 and fibronectin.22Platelet-endothelial-(PE) CAM-1, a member of the Ig superfamily of cell adhesion molecules, and L-selectin are expressed on progenitors.23,24 Finally, several sialomucins have been found on progenitors, including the stem cell antigen, CD34,25 CD43,26,27 CD45RA,28P-selectin glycoprotein ligand-1 (PSGL-1),29 and CD164, described by Zanettino et al30 in this issue ofBLOOD.³¹ Other members from this family not expressed on hematopoietic cells include glycosylation-dependent cell-adhesion molecule-1 (Glycam-1)32 and mucosal addressin cell adhesion molecule-1 (Madcam-1).33 

Which of these receptors is responsible for the specific retention of stem and progenitor cells in the marrow microenvironment or the homing of progenitors to the marrow is not known. In vitro, adhesion of CD34⁺ cells to stromal feeders can be blocked by antibodies against the majority of these receptors.17-24,30,31 In vivo experiments have suggested a relatively predominant role of β1-integrins in the retention of progenitors in the marrow and progenitor homing to the marrow.34-36 However, β1-integrins and their ligands are ubiquitously expressed and β1-integrins cannot provide for the rather exclusive progenitor-marrow interactions. Thus, another receptor(s) must be responsible for the specific stem cell-marrow interactions. The mucin receptor, CD164, described by Zanettino et al,30 or similar—as yet to be characterized—glycoprotein receptors may provide such specificity. In contrast to integrins, whose structure is identical on all cell types, a number of splice variants of CD164 exist, some of which are expressed on endothelial cells but not on hematopoietic cells.30,31 Differences in the glycosylation pattern of CD164 or like molecules may provide further specificity to the receptor.37,38 It has long been speculated that homing of stem cells to the marrow microenvironment depends on lectin-mediated interactions.4 If the sialomucin, CD164, is such a homing receptor, will need to be shown by in vivo transplantation of progenitors from CD164^−/− animals.

Besides being responsible for anatomical localization, adhesion receptors can also transmit signals that modulate the response of the cell to other extracellular factors or directly induce or inhibit cell proliferation, survival, and differentiation. In other biological systems, outside-in signals through integrins and selectins have been most extensively studied.39-42 Engagement of these receptors activates a number of signaling pathways involved with cell proliferation, survival, and differentiation. In the hematopoietic system, coculture of progenitors with stromal feeders inhibits proliferation of progenitors.12-14 The exact mechanism underlying this observation is still unknown. A number of studies have shown that engagement of integrins,14,43-45selectins,46 and, now, mucins31 has profound effects on progenitor survival and growth and may account for the observed contact mediated effects on progenitor proliferation, differentiation, and survival. For instance, when integrins are engaged on CD34⁺ cells cultured with pharmacological concentrations of cytokines, significantly more quiescent progenitors are recruited in cell cycle.44 In contrast, when CD34⁺ cells are cultured under low, more physiological cytokine conditions, engagement of integrins prevents progression of CD34⁺ cells from the G₁ to S phase of the cell cycle, therefore inhibiting progenitor proliferation.14,43 Finally, integrin engagement on CD34⁺ cells cultured in the absence of cytokines or serum serves as a survival signal.45 These studies illustrate that integrin-engagement can affect survival and growth of hematopoietic progenitors. Second, these studies illustrate that regulation of hematopoiesis may be the result of a combined effect of cytokines and contact mediated interactions.

Integrins do not display an enzymatic activity that might serve to trigger a cellular response.47 Signaling requires that the cytoplasmic tail of the integrin colocalizes with the cellular cytoskeleton, which then recruits and activates a tyrosine kinase such as focal adhesion kinase (Fak).48 This leads to activation of Ras, which affects cell proliferation; activation of phosphoinositol-3 kinase (PI-3K), which affects cell proliferation and survival; and alterations in the levels of cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors.49-51 Which of these pathways are involved in integrin-mediated regulation of hematopoietic progenitor proliferation, differentiation, and survival is not yet clear.

In the accompanying report, Zanettino et al30 present evidence that engagement of the mucin, CD164, on normal CD34⁺ cells prevents recruitment of quiescent hematopoietic progenitors into cell cycle when stimulated with a cocktail of cytokines. Engagement of this receptor on CD34⁺CD38⁻ cells induces apoptotic cell death in a large proportion of these cells. The effect of CD164 engagement under different cytokine conditions has not been tested. Because the natural ligand for CD164 is unknown, CD164 receptors were engaged with adhesion blocking anti-CD164 antibodies, a method commonly used in integrin-mediated signaling studies.

Another example of mucin-mediated signaling in hematopoietic cells is prevention of terminal differentiation of myeloid cells seen after enforced expression of CD34,52 an effect dependent on the cytoplasmic tail of the molecule. Consistent with these in vitro studies demonstrating a role for CD34 in prevention of terminal differentiation is the finding that the progenitor population in yolk sac as well as in marrow or spleen of CD34^−/−mice is significantly decreased,53 possibly as a result of premature terminal differentiation.

The signal pathways activated by mucin receptors are yet to be identified. As for integrin receptors, the cytoplasmic tails of mucin receptors do not have intrinsic kinase or other enzymatic function. However, mucins may interact with the cell cytoskeleton through the actin-binding proteins, ezrin and moesin.54 The effect of mucin receptor engagement on intracellular signals has been studied in a number of different cell types, but not yet in hematopoietic progenitor and stem cells. Stimulation of mucin receptors may activate protein tyrosine kinase, the phospholipase C/phosphoinositides signaling pathways, and G-protein signal pathways.55-57Which signal pathways cause cell death or inhibition of cell proliferation or differentiation after stimulation of the CD164 receptor, or other sialomucin receptors, on hematopoietic cells still needs to be determined.

The work describing the cloning and initial functional analysis of a novel adhesion receptor expressed on hematopoietic progenitors illustrates the fact that the hematopoietic process, like other complex biological systems, is governed not only by signals from classically defined cytokines and growth factors, but also by other interactions between the hematopoietic cell and its microenvironment. These cell-cell and cell-ECM contact interactions are responsible not only for the localization of hematopoietic progenitors in the marrow, but also play an important role, like cytokine receptors, in the regulation of progenitor proliferation. Future studies characterizing the signals emanating from adhesion receptors on hematopoietic progenitors will lead to a better insight in the mechanisms that govern the tightly regulated process of normal hematopoietic cell proliferation and differentiation. Such insights may then lead to improved methods for ex vivo manipulation of hematopoietic stem and progenitor cells. There is already evidence that aberrant adhesive interactions caused by decreased function58 or expression59 60 of certain adhesion receptors may underlie the premature mobilization of progenitors in the peripheral blood in certain leukemias and that these defects may in part cause the deregulated proliferation and differentiation seen in leukemic transformation. Future efforts by basic biologists and clinicians are needed to identify potential abnormalities in contact-mediated regulation of progenitor proliferation and differentiation in pathological conditions characterized by increased (leukemias) or decreased (hypoplasia) progenitor growth. This may lead to a better understanding of the pathogenesis of these diseases and new therapeutic approaches for these disorders.