Molecular regulation of hematopoiesis

The molecular nature of stem cell regulatory pathways has been determined using a variety of genetic approaches, including genetic loss-of-function and gain-of-function studies. These have provided several important concepts regarding the molecular control of hematopoiesis. First, some genes have binary functions and are either on or off in various biological states, while other genes function in a continuum and have different effects at different levels. Secondly, while perturbations in single genes may have dramatic cellular effects, cell cycle and lineage effects result from the combinatorial interplay of multiple genes and require coordinated expression of genes with both stimulatory and inhibitory functions. Finally, signal integration often depends on the assembly of large signaling complexes and the spatial proximity of molecules to facilitate interaction is therefore important.

Cell-intrinsic regulators of hematopoiesis

Cell cycle control

The quiescent nature of HSCs is supported by their low level of staining with DNA and RNA nucleic acid dyes, which is consistent with low metabolic activity. Various studies have sought to determine the cell-intrinsic regulators of hematopoiesis involved in HSC cycle control.

Single-cell reverse transcriptase-PCR has been used to profile pertinent transcription factors and other molecules in HSCs induced to differentiate along various lineages by the application of cytokines. This technique has demonstrated the presence of elevated levels of cyclin-dependent kinase inhibitors (CDKIs), suggesting that CDKIs present in HSCs function to exert a dominant inhibitory tone on HSC cell cycling. The bone marrow of mice deficient in CDKI p21aP1/waf1 have increased numbers of HSCs and increased HSC cell cycling, suggesting that p21 functions as a dominant negative regulator of HSC proliferation, a function also noted for other CDKIs, such as p27kip, a negative regulator of hematopoietic progenitor cells.

Self-renewal, commitment, and lineage determination

Many molecules suspected of having importance in stem cell biology have been identified in other systems, such as leukemia, and have encoded transcription factors. Thus, a major focus of molecular research in stem cell regulatory components has involved transcription factors.

Experimental results involving transcription factors have demonstrated cell-intrinsic roles in both global and lineage-specific hematopoietic development. Loss-of-function studies involving the transcription factors c-Myb, AML1 (CBF2), SCL (tal-1), LMO2 (Rbtn2), GATA-2 and TEL/ETV6 have demonstrated global effects on all hematopoietic lineages. Stem cells in animals deficient in these molecules fail to establish definitive hematopoiesis. To test the role of these genes in established hematopoiesis, a method of altering gene expression in the adult animal is required. A molecular technique to address this involves generating conditional knockouts. In these systems, transgenic animals are generated by swapping the wild-type gene of interest with a gene flanked at both ends with lox-p sites, target sites for Cre-recombinase. Such animals can then be mated with transgenic animals expressing the Cre-recombinase driven by different gene promotors. The Cre-recombinase can then be used to specifically excise the gene of interest in a global-, tissue- or developmental-specific manner, depending on the promoter driving the expression of the Cre gene. This approach is technically somewhat limited by the absence of stem cell-specific promoters thus far. However, this approach has aided the identification of critical roles for genes such as Notch-1, which are required for T-cell lineage induction, as Notch-1-deficient mice die during embryogenesis because of a requirement for the protein in other tissues. An interferon-inducible promotor (Mx-Cre) can also be used to turn on Cre-recombinase at specific times by injecting animals with nucleotides, a means of inducing endogenous interferon. This approach has been useful in defining a very different role for SCL in maintaining hematopoiesis in the adult than in establishing it in the developing fetus. This gene product is absolutely required for establishing HSCs. Unexpectedly, there is not a requirement for SCL once the stem cell pool is present in the adult. Rather, SCL is required only for erythroid and megakaryocytic homeostasis. Therefore, transcription factor regulation of the stem cell compartment is highly dependent on the stage of development of the organism. Lineage-specific effects of transcription factors may also be stage-dependent.

Loss-of-function studies have also proved useful in identifying lineage-specific transcription factors. Mice genetically deficient in the transcription factor Ikaros lack T and B lymphocytes and natural killer cells, but maintain erythropoiesis and myelopoiesis. Mice lacking the ets-family transcription factor PU.1 demonstrate embryonic lethality. However, mutant embryos produce normal numbers of megakaryocytes and erythroid progenitors but have impaired erythroblast maturation and defective generation of progenitors for B and T lymphocytes, monocytes and granulocytes. While the outcome of such genetic lesions can be assessed, it remains unclear whether such lesions result in failure to establish a commitment program or the execution of an established program.

Gain-of-function studies have been used similarly to assess the roles of various global and lineage-specific transcription factors. Enforced expression of the HoxB-4 homeobox gene in HSCs confers heightened capacity for in vivo stem cell function. Similarly, ectopic expression of HoxB4 in embryonic stem cells combined with in vitro culture on stroma induces a switch to the definitive hematopoiesis phenotype that is transplantable into adult recipients. Mice deficient in the Pax-5 transcription factor suffer from severe impairment of the B-lymphoid lineage. This phenotype may be rescued by reintroduction of wild-type Pax-5.

In alternative model systems, lineage reprogramming may be achieved by ectopic expression of transcription factors. Introduction of the erythrocytic lineage transcription factor GATA-1 reprograms avian myeloblast cells down eosinophilic and thromboblastic lineages. Introduction of the dominant negative retinoic acid receptor-alpha (RARa) into murine stem cells permits the establishment of permanent cell lines that grow in response to stem cell factor and maintain the ability to differentiate along myeloid, erythroid and B-lineage lines. The points in the hematopoietic cascade at which specific transcription factors play a role are illustrated in Figure 3.3.

LT-HSC

ST-HSC

LT-HSC

ST-HSC

SCL (++) C/EBPa (±) GATA-2 (++) PU.1 (±) NF-E2 (-) Aiolos (±) GATA-1 (±) GATA-3 (±)

Fig. 3.3 Transcription factors active at various stages of hematopoiesis. Redrawn with permission from Nature (2000), 404, p.196

Lymphoid pathway CLP

Lymphoid pathway CLP

Myeloid pathway CMP

Myeloid pathway CMP

Pro-T

Pro-T

T cell

Pro-B

Pro-B

B cell

Monocyte Granulocyte

Megakaryocyte Erythrocyte

Cell-extrinsic regulators

Ultimately, hematopoietic stem and progenitor cell decisions are regulated by the coordinated action of transcription factors as modified by extracellular signals. Extracellular signals in the form of hematopoietic growth factors are mediated via cell surface hematopoietic growth factor receptors. He-matopoietic growth factors exert specific effects when acting alone and may have different effects when combined with other cytokines. There are at least six receptor superfamilies, and most growth factors are members of the type I cytokine receptor family. The effects of various cytokines during my-elopoiesis are illustrated in Figure 3.4.

Type I cytokine receptors

Type I receptors do not possess intrinsic kinase activity but lead to phosphorylation of cellular substrates by serving as docking sites for adapter molecules with kinase activity. Examples of receptors in this family include leukemia inhibitory factor (LIF), interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-18, GM-CSF, G-CSF, erythropoietin, prolactin, growth hormone, ciliary neurotrophic factor and c-mpl. These receptors share several features, including enhanced binding and/or signal transduction when expressed as hetero-or homodimers, four cysteine residues and fibronectin type III domains in the extracellular domain, WSXWS ligand-binding

HPP-CFC

Mixed progenitor_ cell

CFU-GEMM

HPP-CFC

CFU-GEMM

Pluripotent stem cell

Mixed progenitor_ cell

G-CSF GM-CSF I IL-4 t Myeloblast

IL-5 GM-CSF

Myelomonocytic progenitor

Monoblast

Monoblast

CSF-1 GM-CSF

Promonocyte

CSF-1

Monocyte

G-CSF GM-CSF I IL-4 t Myeloblast

G-CSF GM-CSF

Myelocyte

G-CSF

Neutrophil

IL-5 GM-CSF

Eosinophil

Chemokines

Pluripotent stem cell

Basophil, mast cell

Fig. 3.4 Cytokines active at various stages of hematopoiesis. Modified from Figure 16.3 in Hematology: Basic Principles and Practice, 3rd Edition, Ronald Hoffman, ed., 2000, with permission from Elsevier sequence in the extracellular cytokine receptor domains, and lack of a known catalytic domain in the cytoplasmic portion. Another shared feature of receptors in this family is the ability to transduce signals that prevent programmed cell death (apoptosis).

Several exceptions to this family with intrinsic kinase activity are the hematopoietic growth factor receptors platelet-derived growth factor receptor (PDGFR), flt-3 receptor and c-fms, which are the ligands for steel factor (SF), Flt-ligand (FL) and M-CSF, respectively (Table 3.1).

Type II cytokine receptors

This class includes the receptors for tissue factor, IL-10 and interferon y (IFN-y). This family contains a type III fibronec-tin domain in the extracellular domain, like the type I family.

Protein serine-threonine kinase receptors

This family includes the 30 members of the transforming growth factor-P (TGF-P) superfamily, which bind to their receptors as homodimers. Members of this family include the three TGF-P receptors: type I (TbRI, 53 kDa), type II (TbRII, 75 kDa) and type III (TbRIII, 200 kDa). Members of this family have a profound inhibitory effect on the growth and differentiation of hematopoietic cells and on auxiliary hematopoietic cells. Binding of TFG-P requires TbRII. After binding, signal transduction occurs via activation of serine-threonine kinase cytoplasmic domains of the receptor chains, which results in the phosphorylation of Smad molecules on serines. Phosphorylated Smad complexes translocate to the nucleus, where they induce or repress gene transcription. TGF-P is the best characterized negative regulator of hematopoiesis. It inhibits mitosis by inducing cell cycle inhibitors such as p21ap1/ waf1, p27kip1 and p16INK4a, inhibiting the cyclin-dependent kinases Cdk4 and Cdk6, and inducing phosphorylation of the retinoblastoma protein. The TGF-P receptor family and its downstream mediators act as braking factors for a number of cell types and are frequently inactivated by somatic mutation in a number of cancers.

Chemokine receptors

This family comprises seven transmembrane-spanning G-protein-coupled receptors that influence both cell cycle and cellular movement, or chemotaxis. These receptors are divided into three families, a or CXC, P or CC, and y or C, on the basis of variability in cysteine residues. The best characterized is CXCR4, which mediates homing and engraftment of HSCs in bone marrow and is critical to hematopoietic development. IL-8 and Mip-1a act as inhibitors of progenitor cell prolifera tion. Members of this receptor family have also been implicated in cancer metastasis and the entry of HIV-1 into cells.

Tumor necrosis factor receptor family

Members of the tumor necrosis factor receptor (TNFR) family have varied effects, some having the ability to induce programmed cell death and others stimulating mesenchymal cells to secrete hematopoietic growth factors. These receptors contain Cys-rich extracellular domains and 80-amino acid cytoplasmic 'death domains', which are required for transducing the apoptotic signal and inducing NF-kB activation. Members of this family include TNFR1, TNFR2, fas, CD40, nerve growth factor (NGF) receptor, CD27, CD30 and 0X40, each with at least one distinct biological effect.

Components of the hematopoietic microenvironmental niche

While soluble factors influence stem cell fate, these factors are seen by the cell in the context of cell-cell contact among heterologous cell types and cell-matrix contact, which make up the three-dimensional setting of the bone marrow. What actually constitutes the critical microenvironment for hematopoiesis is surprisingly poorly defined. The ability of primitive cells to mature in vitro in complex stromal cultures suggests that at least some elements of the regulatory milieu of the bone marrow can be recapitulated ex vivo. Studies based solely on ex vivo systems are suspect, however, as no fully satisfactory recreation of stem cell expansion or self-renewal has been defined. Recognizing this limitation, it has been determined that mesodermal cells of multiple types are needed to enable hematopoietic support. These include adipocytes, fibroblastic cells and endothelium. Recently, in vivo studies have indicated that the osteoblast may perform a key regulatory role in stem cell self-renewal, and the activation of this cell can affect the number of stem cells.

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