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266 Part IV: Molecular and Cellular Hematology Chapter 18: Hematopoietic Stem Cells, Progenitors, and Cytokines 267

hematopoietic microenvironment, and has profound effects on HSC HSCs on its introduction into these cells, and whose genetic elimina-
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localization to the stem cell niche. However, this chemokine is also tion leads to a profound deficit in numbers of HSC in vivo. Although
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thought to display direct effects on the survival and proliferation of the hormone fails to affect total cellular levels of HOXA9 in either
hematopoietic stem and progenitor cells, both alone and in synergy model cells or primary primitive murine HSC populations, TPO greatly
with other hematopoietic cytokines. 208,209 enhances HOXA9 nuclear translocation by inducing expression of its
Notch Ligands The human homologue of Drosophila Notch was translocation partner, MEIS1, and leading to ERK1/2 MAPK-induced
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identified as an altered gene product in T-cell leukemia. The discov- MEIS1 phosphorylation. 231
ery that the hematopoietic microenvironment displays Notch ligands, A third mechanism by which cytokines affect HSC expansion is
and that Notch isoforms appear on primitive hematopoietic cells, 211,212 through global inhibitors of signaling. In addition to its direct effects on
opened the possibility that Notch affects HSCs. This assertion has been HSC survival and self-renewal pathways, TPO has been shown to inter-
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directly proven: The Notch ligands Delta1 and Delta4 expand primitive act with the adaptor protein LNK, which inhibits signaling pathways
hematopoietic cells. 213,214 It is possible that the favorable effect of marrow derived from a broad range of hematopoietic cytokines, 233,234 including
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osteoblasts on HSCs is a result of their expression of Notch ligands, as TPO. From these data it appears that TPO and LNK alternately regu-
inhibition of Notch processing blocks the expansion in HSCs seen in late HSC expansion and each other. 236
mice in which osteoblasts have been experimentally expanded. 158
Wnt Proteins A role for Wnt proteins in hematopoiesis was sug- MATRIX PROTEINS
gested by their localization at sites of fetal blood cell production and
their ability to expand hematopoietic progenitor cells. Wnt 3a has Fibronectin
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been shown to expand long-term repopulating HSCs. 216,217 As Wnt pro- FN is a 450-kDa fibril-forming glycoprotein composed of two subunits
teins are expressed on primitive hematopoietic cells, it is also possible that is a major component of the hematopoietic microenvironment. FN
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that in addition to classical paracrine signaling, Wnts could act in an is produced by both marrow stromal (endothelial cells and fibroblasts)
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autocrine fashion in HSC biology. and blood cells, and is implicated in marrow homing of hematopoi-
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Transforming Growth Factor β The transforming growth factor etic cells. Distinct domains of FN have been identified that interact
(TGF) family of ligands (TGF-β, activins, bone morphogenetic pro- with different integrins, for example, those for integrin α β and for
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teins [BMP]) bind to members of the TGF-β receptor family and trig- integrin α β . HSCs display multiple integrins and their engagement
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1
ger activation of the SMAD (Sma- and Mad-related protein) group of contributes to cell survival and/or expansion. For example, ex vivo cul-
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intracellular mediators. Unlike the cytokines discussed above, TGF-β ture of human CD34+ cells on FN maintains the repopulating capac-
members inhibit HSC cycling, 220,221 and so blunt cell expansion, at least ity of HSCs, whereas growing the cells in suspension obliterates their
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in vitro. Nevertheless, the situation in vivo is complex; genetic elimina- ability to repopulate hematopoiesis. FN binding to α β integrins also
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tion of TGF-β does not alter HSC self-renewal or regeneration in vivo, enhances the generation of large numbers of committed hematopoi-
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likely because of redundancy in the TGF-β system of ligands. In con- etic progenitors and LTC-IC from primitive precursors. Multiple
trast, genetic elimination of several of the SMAD proteins disrupts nor- molecular mechanisms for the effects of FN on integrin-bearing cells
mal HSC homeostasis. 224,225 Recent data suggests that BMP4 might be have been identified, and serve as a paradigm for the supportive effects
the critical member of the TGF family that affects HSC biology. 226 of this entire class of microenvironmental signals.
The mechanisms by which these cytokines exert their effects on Integrin engagement by FN triggers a number of intracellular sig-
HSCs are only now beginning to be understood at the molecular level, naling events that affect the cellular cytoskeleton and transcriptional
but it is already clear that effects on the transcription factors that govern events. Complexes composed of kinases, adaptors, and cytoskeletal
HSC survival, self-renewal, and expansion likely play critical roles. It components are recruited to sites of integrin engagement, initiated by
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has long been understood that Wnt proteins act to stimulate an increase interactions with integrin cytoplasmic domains. A critical molecule
in intracellular levels of β-catenin, a nascent transcription factor. Upon for integrin-based signaling is paxillin, a 68-kDa protein that contains
being liberated from proteasomal degradation in the presence of Wnt, a number of protein–protein binding domains, and which binds to the
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β-catenin translocates to the nucleus and alters transcription of genes cytoplasmic domain of the integrin. Additional binding partners also
displaying the T-cell factor (TCF)/lymphoid-enhancer binding factor help trigger intracellular signaling, including focal adhesion kinase
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(LEF) consensus sequence. Moreover, TGF-β–induced alterations (FAK) and the closely related Pyk2 kinase. Upon recruitment, FAK
in SMAD protein phosphorylation affects their ability to activate tran- and Pyk2 are activated and initiate Tyr phosphorylation of paxillin and
scription directly. However, most of the cytokine receptors that affect other associated molecules, creating additional protein binding sites
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HSCs do not directly affect transcription factors; rather, several cytok- and activating tethered secondary messenger molecules. One vital sig-
ines affect signaling pathways that alter the expression, activity, or sub- naling pathway downstream of FAK and Pyk2 is PI3K, which is medi-
cellular localization of HSC transcription factors. ated by the association of its regulatory p85 subunit with the adhesion
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As discussed in “Hematopoietic Stem Cell to Common Myeloid kinases (Chap. 14). FAK also directly activates a pathway that results
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Progenitor Commitment” above, SCL is a helix-loop-helix transcription in upregulation of the cyclin D promoter, affecting cell proliferation.
factor critical for hematopoiesis. SCF enhances the survival of primi- Integrin engagement also leads to Src activation, engagement of Grb2,
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tive hematopoietic cells in culture by maintaining their expression of and activation of Ras, pathways also activated by SCF and TPO, and
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SCL, which enhances expression of the SCF receptor c-Kit. Two potentially providing a mechanism by which diverse extrinsic stimuli of
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additional transcription factors that play vital roles in HSC expansion, HSCs may converge.
HOXB4 and HOXA9, are both affected by cytokines. Exogenous expres-
sion of HOXB4 to levels only twice normal are associated with a marked Hyaluronan
and rapid expansion of transduced HSCs on their transplantation into Another stromal cell matrix glycoprotein is hyaluronan, which binds
lethally irradiated recipients. In both model cell lines and primitive to two hematopoietic cell-surface receptors, RHAMM and CD44.
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hematopoietic cells TPO doubles the expression of HOXB4, in a p38 Although most CD34+ marrow cells express CD44, only a fraction of
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MAPK-dependent fashion. Of probably greater significance is the them adhere to hyaluronan, a process that can be mediated by cytok-
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effect of TPO on HOXA9, a gene that also induces rapid expansion of ines, as a result of either increased surface expression of CD44 or an
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