TIBS 17 - AUGUST 1992
THE FIRSTHEMOPOIETICREGULATOR, erythropoietin, was discovered in 1906 through some simple animal experiments, but there was no further progress until the mid 1960s when semisolid culture techniques were developed that could support the growth of various types of hemopoietic colonies from normal bone marrow cells 1,2. These cultures not only demonstrated that hemopoietic cells cannot divide in the absence of added stimulating factors, but also provided convenient bioassays for the active factors present in various tissue extracts or in media conditioned by such tissues. In the 1970s separative protein chemistry techniques were used to purify hemopoietic regulators from natural tissue sources. However, this proved to be technically demanding due to the very low concentrations of hemopoietic regulators in even the richest sources. Final success required complex sequential separative procedures and, above all, application of the evolving techniques of high-performance liquid chromatography (HPLC). By 1983, erythropoietin, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), multipotential colony stimulating factor (Multi-CSF/IL3), interleukin 2 (IL-2) and IL-1 had all been purified, but these were obtained in minute quantities and were available only for the laboratories involved. While some interesting cell biology was possible using this material, the amounts produced were so low that testing in vivo was impossible, even in small laboratory animals. In retrospect, this period was the turning point in the development of these regulators. Between 1984 and 1986, cDNAs encoding erythropoietin and the four CSFs were isolated 3-9, and active recombinant factors were mass produced using bacterial, yeast or mammalian expression systems. These recombinant factors, when administered to animals, selectively stimulated the formation of the particular hemopoietic cells that were known to be responsive to the factors from culture studies I°. Corresponding huma6 cDNAs D. Metcalf is at The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, 3050 Victoria, Australia.
286
Hemopoietic regulators The production and maturation of blood cells from the eight major blood cell lineages is a complex and continuous process, which is largely controlled by specific glycoprotein hemopoietic regulators. These regulators also control the functional activity of the blood cells through eliciting a diverse set of intracellular responses initiated by a regulator-specific membrane receptor. Twenty of these regulators have now been characterized, and their mass production has led to four already being licensed for clinical use in disease states involving subnormal blood cell formation.
were also isolated during this period, leading to successful primate preclinical trials and the first human trials in patients with anemia or low white cell levels. The clinical effectiveness and low toxicity of erythropoietin, GM-CSF and G-CSF allowed their rapid licensing for clinical use. These three agents are regarded by many as the first commercially successful products of molecular biology applicable in clinical medicine. The following years have seen the characterization of additional regulators at an ever-increasing rate (Table 1). The progressive use of direct cDNA ex-
pression cloning to detect these factors has resulted in a somewhat disconcerting situation: purified recombinant factors have become available without any preceding framework of biological knowledge on their likely role in normal hemopoiesis. From a personal viewpoint as a worker on the CSFs, I think the discoveries in the 1984-1992 period provided three pieces of information that were crucial for the development of the field. First, the demonstration that genes encoding CSFs exist in the mammalian genome 3-9 removed the lingering doubt that the
Table I. The hemopoietic regulators Regulator(Abbreviation)
Respondinghemopoieticcells
Erythropoietin(Epo) Granulocyte-macrophagecolonystimulatingfactor (GM~SF) Granulocytecolonystimulatingfactor (G-CSF) Macrophagecolonystimulating factor (M-CSF) Multipotential colonystimulatingfactor (Multi-CSF/IL-3) Interleukin 1 (IL-1) Interleukin 2 (11.-2) Interleukin 4 (IL-4) Interleukin 5 (IL-5) Interleukin 6 (IL-6) Interleukin 7 (IL-7) Interleukin 9 (11.-9) Interleukin 10 (IL-IO) Interleukin 11 (IL-11) Interleukin 12 (IL-12) Megakaryocytecolonystimulating factor (Meg~SF) Stem cell factor (SCF) Leukemia inhibitoryfactor (LIF) Oncostatin M (OSM) Macrophageinflammatory protein c((MIP-I(~)
E,Meg G,M,Eo,Meg,E G,M M,G G,M,Eo,Meg,Mast,E,Stem " T,Stem T,B B,T,G,M,Mast Eo,B B,G,Stem,Meg B,T T,Meg,Mast T Meg,B NK Meg Stem,G,E,Meg,Mast Meg ? Stem
Abbreviations: G, granulocytes;M, macrophages;Eo, eosinophils; E, erythroidcells; M, megakaryocytes; Stem, stem cells; Mast, mast cells; T, T lymphocytes;B, B lymphocytes;NK, natural killer cells.
© 1992,ElsevierSciencePublishers,(U!O
TIBS 1 7 -
AUGUST1992
painfully purified native CSFs might merely have been products of contaminant microorganisms in the tissues used as source materials. Second, the demonstration that CSFs stimulate granulocyte and macrophage formation in vivo ]° meant that the preceding 20 years of culture work had not merely been documenting some trivial, if spectacular, in vitro artifacts. Third, the demonstration that osteopetrotic (op/op) mice with severe defects in osteoclast and macrophage formation have an abnormality in their M-CSF-encoding gene that prevents transcription of M-CSF mRNA, and that the disease is correctable by the injection of M-CSFu. This provided proof that at least one CSF functions in vivo as a genuine regulator important for normal hemopoiesis.
(a)
(b)
G.cse
--°\1 7 s/~-
GM Progenitor
(c) G-CSF
G-CSF
1
~
. M-CSF ~''L-O
G-CSF
1 GM Progenitor cell
Myeloblast
1~
~roxlckD
I~'mgooytoW~
©©© Granulocytes
©© Granulocyte,*
© Macroph,,ge8
Maturo Grenulocyte
Rgure 1 Key aspects of the biology of hemopoietic regulators as exemplified by the control of granulocyte formation and function. (a) Cell production in any one lineage is dependent on regulator stimulation, but multiple regulators can directly stimulate cell proliferation because of co-expression of multiple sets of receptors on individual cells. Regulators have multiple additional actions including (b) the induction of commitment to form cells in a restricted lineage, (c) initiation of the complex events of maturation and (d) stimulation of the functional activity of the mature progeny that are finally produced.
are low (200-500 per cell) and only a small proportion of these needs to be Most of the hemopoietic regulators occupied to stimulate proliferation ~3. are glycoproteins with polypeptide Simultaneous stimulation by two or molecular masses of 14-21 kDa. Despite more relevant growth factors elicits enthis general similarity, and evidence of hanced proliferative responses. While shared functional activity, there ap- more mature precursors (usually compears to be little amino acid sequence mitted to a single lineage) can respond homology between the regulators. to stimulation by single growth factors, However, it is becoming increasingly the least mature hemopoietic cells likely that many exhibit comparable (stem cells) require combined sigthree-dimensional conformations in- nalling from multiple factors before the volving four ¢z-helical bundles, in which onset of proliferation. two helices combine to produce the The CSFs were the first regulators to reveal clearly that growth factors not active binding domain ~2. The CSFs are prototype examples of only control cell proliferation but can the mandatory role played by positive also regulate differentiation commithemopoietic regulators in controlling ment, maturation induction and the hemopoietic cell division. They can act functional activity of mature post-mitotic at concentrations of less than 1 ng ml-~ cells finally produced (Fig. 1) ~°. This to exert a concentration-dependent di- concept of polyfunctionality is now rect action in controlling cell cycling thought to apply to many, perhaps all, status, the length of the cell cycle and growth factors. With some, such as IL-6~4 the total number of progeny produced or leukemia inhibitory factor (LIF)~5, by individual precursor cells ~°. polyfunctionality also includes the The hemopoietic regulators exhibit ability to act on a wide range of nonapparent redundancy in their actions hemopoietic target cells. For example, on hemopoietic cells. For cells in any LIF also acts on osteoblasts, neurones, one lineage, there are multiple regu- hepatocytes and adipocytes. What uselators that can stimulate proliferation, ful purpose could be served in the body due to simultaneous expression of by such polyfunctional regulators? In specific receptors for the different regu- part, unwanted responses may be minilators on individual cells. Typically, mized by local production of the regureceptor numbers for any one regulator lator, resulting in effects limited to ad-
The regulators and their actions
(d)
jacent responding cells, but the question as such remains unanswered.
The receptors for hemopoietic regulators The overlapping actions of the different regulators and their polyfunctionality pose problems, since there appears to be only a single species of non-cross-reactive membrane receptor for each regulator. In the past three years an explosion of information on the receptors for hemopoietic growth factors has resolved some of these dilemmas. Some hemopoietic regulators, such as stem cell factor (SCF)]~ or M-CSF~7have classical transmembrane tyrosine kinase receptors, but the large majority of hemopoietic regulators have glycoprotein transmembrane receptors that lack a tyrosine kinase domain TM. These are now recognized to form a new group of growth factor or cytokine receptors that exhibit significant homology in their extracellular domain. Furthermore, the low-affinity tt-chains of these receptors can be converted to high-affinity receptors by association with a ~subunit. It is believed to be the ~-subunit that then initiates signalling from the ligand-receptor complex (Fig. 2). On human cells, a common ~-subunit combines competitively with the it-chains of GM-CSF, Multi-CSF or IL-5,
287
TIBS 17 - A U G U S T
which explains certain competitive interactions between the three regulators and why all ~ r e e share a common capacity to stimulate eosinophil proliferation 19. The obvious relatedness of the a-chain receptors for hemopoietic regulators indicates that they may derive from a common ancestral receptor, providing the most convincing single piece of evidence for the common evolutionary origin of many of the hemopoietic regulators. This work has now raised the further possibility that the multiple actions of individual CSFs may be mediated by multiple subunits, each initiating distinct signalling cascades that impinge on various targets within the cell.
1992
poietic regulators owing to the ability of these factors to stimulate the functional activity of mature cells. This is seen most clearly with macrophage stimulating factors such as GM-CSF. In GMCSF transgenic mice that have consistently elevated levels of GM-C~F, excess stimulation results in the production of toxic products such as tumor necrosis factor, IL-1 or plasminogen activator by the macrophages in vivo. This can lead to localized inflammatory disease and general tissue damage 23. The possible role of overproduction of hemopoietic regulators in the development of comparable disease states in humans remains to be clearly established.
0£
The future Clinical applications The hemopoietic regulators have been proven clinically to be effective in stimulating the formation of mature blood cells; erythropoietin is now the treatment of choice in the anemia of chronic renal disease 2°. This situation is comparable to the use of insulin in diabetes where the insulin-producing cells in the pancreas have been destroyed. The biology of erythropoietin production in the adult is not typical of hemopoietic regulators, in that most erythropoietin is produced in a single organ, the kidney, and erythropoietinproducing cells are damaged in renal disease. The CSFs can stimulate production of granulocytes and monocytes in disease states where production of these cells is subnormal 21,22,a situation common in cancer patients following chemotherapy or marrow transplantation. CSF production is more complex than for erythropoietin, since the CSFs originate from multiple cell types dispersed throughout the body. In healthy subjects, there are only very low levels of CSFs, but production is strongly induced by endotoxins and other microorganism products. However, these induced responses do not always result in the production of optimal levels of CSFs, even in severe aplasia or infection. This is illustrated by the ability of injected additional CSF to induce enhanced cell formation and functional activity in such patients. Most of the hemopoietic regulators now available in an active recombinant form have yet to enter clinical trials, but it is likely that at least some will be valuable in correcting hemopoietic defects, accelerating the regeneration of hemopoietic tissues after injury or in
288
Rgure 2 Schematic representation of the human membrane receptor for GM-CSF. The unique c¢-subunit contains a motif, in common with other cytokine receptors, that has spaced Cys residues (CCCC) and a Trp-Ser motif (WS). The (z-chain binds GM-CSF with low affinity, possibly in a crevice between the tandem ~barrels (stippled regions). A high-affinity receptor is then generated by crosslinking with the ~subunit, which itself contains two repeats of the cytokine receptor motif. Intracellular signalling may then be initiated by the intracytoplasmic domain of the ~subunit.
enhancing the functional activity of existing cells.
Overstimulation by hemopoietic regulators Chronic stimulation by excessive concentrations of hemopoietic regulators can lead to extreme hyperplasia in responding hemopoietic populations but is not sufficient alone to induce leukemic transformationz3-25. However, if cells acquire the abnormal ability to produce a growth factor to which they can respond it can contribute to leukemic transformation. If this is in conjunction with an intrinsic abnormality in their self-renewal during cell division, the combined abnormalities certainly lead to leukemic transformation2E Other disease states may develop as a result of excessive stimulation by the hemo-
What is the possibility that the regulatory control of hemopoiesis is typical of what occurs in other tissues? Certainly there are unusual features of hemopoiesis, such as the dispersion of cell-production sites, the complexity of specialized lineage formation from common precursor cells and the need to control mature cells in remote locations. These lend themselves to a regulatory solution that makes use of a complex of specific regulatory molecules. However, in principle, all tissues present similar regulatory problems. It is therefore reasonable to presume that the control of all tissue types is achieved by comparable regulators. The hemopoietic regulators proved relatively easy to discover because of the availability of culture systems supporting the growth and function of appropriate hemopoietic cells. Given the development of culture technology for other cell types, it is virtually certain that a very large number of specific regulators will be detectable through actions on these cells. The 20 hemopoietic regulators isolated so far are but a beginning. Major frontiers in cell biology lie waiting to be broached by the adventurous, for the golden age of regulator discovery is upon us.
References I Bradley,T. R. and Metcalf, D. (1966) Aust. J. Exp. Biol. Med. Sci. 44, 287-300 2 Ichikawa,Y., Pluznik, D. and Sachs, L. (1966) Proc. Natl Acad. Sci. USA 56, 488-495 3 Yokota,T. et al. (1984) Proc. Natl Acad. Sci. USA 81, 1070-1074 4 Fung,M-C.et al. (1984) Nature 307, 233-237 5 Gough,N. M. et al. (1984) Nature 309, 763-767 6 Souza,L. M. et al. (1986) Science 232, 61-65 7 Nagata,S. et al. (1986) Nature 319, 415-418 8 Kawasaki,E. S. et al. (1985) Science 230, 291-296 9 Jacobs, K. et al. (1985) Nature 313, 806-810
TIBS 1 7 - AUGUST1992 10 Metcalf, D. (1991) Philos. Trans. R. Soc. London Ser. B 333, 147-173 11 Wiktor-Jedrzejczak,W. et al. (1990) Proc. Natl Acad. Sci. USA 87, 4828-4832 12 Bazan, J. F. (1990) Immunol. Today 11,
350-354 13 Nicola, N. A. (1989) Annu. Rev. Biochem. 58, 45-77 14 Kishirnoto, T. (1989) Blood 74, 1-10 15 Metcalf, D. (1991) Int. J. Cell Cloning9,
DUCHENNE MUSCULAR DYSTROPHY (DMD) is a common neuromuscular disease that occurs in approximately 1 out of every 3500 live male births 3. Although the disease is present from birth and may cause developmental delays such as late onset of walking, the affected patient usually does not present until three to five years of age. Initial complaints often include leg weakness, which manifests itself in running and stair climbing difficulties and problems with rising from the floor. As the affected child grows, the loss of muscle strength causes increasing convex curvature of the spine (lordosis) and a waddle-like gait when walking. The gradual muscle-wasting generally leads to loss of ambulation by 12 years of age. The progressive loss of muscle continues throughout life with the proximal muscles affected first, followed late~" by the more distal muscles with respiratory failure and death frequently occurring by the late teens or early twenties 3. Becker muscular dystrophy (BMD) is an aUelic disorder that is less common than DMD, affecting 1 in 30 000 live male births 4. The course of BMD is much more variable and less severe than that of DMD for example, many Becker patients remain ambulatory well into adulthood and may live full and minimally restricted lives 3. At the cellular level, both DMD and BMD involve the loss of individual muscle fibers. The usual arrangement of muscle fibers is disrupted and there is marked degeneration, regeneration and fibrosis in the muscles (Fig. 1)3. While there are some abnormalities that can be detected at birth, fibrosis and fatty replacement become more obvious over time. M. D. S. Anderson and L. M. Kunkel are at
HowardHughesMedical Institute and the Division of Genetics, Children'sHospital, Harvard Medical School, Boston, MA 02115, USA.
95-108 16 Chabot, B. et al. (1988) Nature 335.88-89 17 Sherr, C. J. et al. (1985) Cell 41, 665-676 18 Bazan, J. F. (1990) Proc. Natl Acad. Sci. USA
87, 6934-6938 19 Nicola, N. A. and Metcalf, D. (1991) Cell 67,
1-4 20 Eschbach, J. W. et al. (1987) New Engl. J. Med.
316, 73-78 21 Glaspy, J. A. and Golde, D. (1990) Exp.
Hematol. 18, 1137-1141 22 Davis, I. and Morstyn, G. (1991) Semin. Hematol. (Suppl.) 2 28, 25-33 23 Lang, R. A. et al. (1987) Cell 51, 675-686 24 Chang, J. M., Metcalf, D., Gonda, T. J. and Johnson, G. R. (1989) J. Lab. Clin. Invest. 84,
1488-1496 25 Chang, J. M. eta/. (1989) Blood 73,
1487-1497 26 Metcalf, D. (1989) Cancer Res. 49, 2305-2311
The molecular and biochemical basis of Duchenne muscular dystrophy
Duchenne and Becker muscular dystrophy (DMD, BMD) have both been clinically recognized for over 100 years, yet throughout much of that time nothing beyond clinical evaluation and supportive care during the disease course was available to patients. The identification of the molecular basis of DMD/BMD in 19861,2 paved the way for extensive progress toward the understanding, diagnosis and treatment of this disease.
The severity and frequency of DMD 427 kDa protein dystrophin. The sheer and BMD led to a great deal of effort in size of the gene is thought to account numerous laboratories to identify the for the unusually high mutation rate at cause of these disorders. The unequal the locus. gender distribution of the diseases gave the initial hint that the genetic location Gene characterization of DMD and BMD would be on the X The entire coding region of the gene chromosome. This was confirmed cyto- has been sequenced, providing extensive logically by analysis of rare manifesting information about the primary struccarrier females 5 and by linkage analysis 6 ture of dystrophin 8, the relationship in the early 1980s. Both types of analy- between the sizes and locations of deses narrowed the location to Xp21, letions within the gene and the clinical paving the way for identification of the presentation of the disease 9. gene itself. The increase in information about The approach used to identify the the enormous gene size has made it DMD/BMD gene set the stage for future clear that mRNA processing plays an research into other human disease- important role in DMD/BMD trancausing genes. The strategy, now scription. For example, there are a numtermed positional cloning, involved the ber of isoforms of dystrophin that differ search for and identification of a dis- at the carboxyl terminus and are proease gene with no prior knowledge of duced by alternative mRNA splicing ]°, the gene product. Based solely on the and there are at least three known map position of mutations causing the alternative transcription start points 11-]3. disease, part of the gene was identified Questions of how accurate splicing by our group in 1986 ], and a different occurs and what regulates alternative section by Ron Worton's group a few splicing of such a large gene will only be months later 2. There then followed a answered with more extensive knowlrapid description of the encoded pro- edge of the non-coding regions of the tein dystrophin 7 and the realization of DMD gene sequence. Until recently, the the complexity of the gene and its pro- examination of DMD gene processing cessing. The 2.5 million-base-pair gene has been thwarted by the enormous consists of at least 70 exons, resulting size and complexity of the DMD gene. in a 14 kb transcript which encodes the Recent construction of yeast artificial
© 1992, Elsevier Science Publishers, (UK) 0376-5067/92/$05.00
289
THE FIRSTHEMOPOIETICREGULATOR, erythropoietin, was discovered in 1906 through some simple animal experiments, but there was no further progress until the mid 1960s when semisolid culture techniques were developed that could support the growth of various types of hemopoietic colonies from normal bone marrow cells 1,2. These cultures not only demonstrated that hemopoietic cells cannot divide in the absence of added stimulating factors, but also provided convenient bioassays for the active factors present in various tissue extracts or in media conditioned by such tissues. In the 1970s separative protein chemistry techniques were used to purify hemopoietic regulators from natural tissue sources. However, this proved to be technically demanding due to the very low concentrations of hemopoietic regulators in even the richest sources. Final success required complex sequential separative procedures and, above all, application of the evolving techniques of high-performance liquid chromatography (HPLC). By 1983, erythropoietin, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), multipotential colony stimulating factor (Multi-CSF/IL3), interleukin 2 (IL-2) and IL-1 had all been purified, but these were obtained in minute quantities and were available only for the laboratories involved. While some interesting cell biology was possible using this material, the amounts produced were so low that testing in vivo was impossible, even in small laboratory animals. In retrospect, this period was the turning point in the development of these regulators. Between 1984 and 1986, cDNAs encoding erythropoietin and the four CSFs were isolated 3-9, and active recombinant factors were mass produced using bacterial, yeast or mammalian expression systems. These recombinant factors, when administered to animals, selectively stimulated the formation of the particular hemopoietic cells that were known to be responsive to the factors from culture studies I°. Corresponding huma6 cDNAs D. Metcalf is at The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, 3050 Victoria, Australia.
286
Hemopoietic regulators The production and maturation of blood cells from the eight major blood cell lineages is a complex and continuous process, which is largely controlled by specific glycoprotein hemopoietic regulators. These regulators also control the functional activity of the blood cells through eliciting a diverse set of intracellular responses initiated by a regulator-specific membrane receptor. Twenty of these regulators have now been characterized, and their mass production has led to four already being licensed for clinical use in disease states involving subnormal blood cell formation.
were also isolated during this period, leading to successful primate preclinical trials and the first human trials in patients with anemia or low white cell levels. The clinical effectiveness and low toxicity of erythropoietin, GM-CSF and G-CSF allowed their rapid licensing for clinical use. These three agents are regarded by many as the first commercially successful products of molecular biology applicable in clinical medicine. The following years have seen the characterization of additional regulators at an ever-increasing rate (Table 1). The progressive use of direct cDNA ex-
pression cloning to detect these factors has resulted in a somewhat disconcerting situation: purified recombinant factors have become available without any preceding framework of biological knowledge on their likely role in normal hemopoiesis. From a personal viewpoint as a worker on the CSFs, I think the discoveries in the 1984-1992 period provided three pieces of information that were crucial for the development of the field. First, the demonstration that genes encoding CSFs exist in the mammalian genome 3-9 removed the lingering doubt that the
Table I. The hemopoietic regulators Regulator(Abbreviation)
Respondinghemopoieticcells
Erythropoietin(Epo) Granulocyte-macrophagecolonystimulatingfactor (GM~SF) Granulocytecolonystimulatingfactor (G-CSF) Macrophagecolonystimulating factor (M-CSF) Multipotential colonystimulatingfactor (Multi-CSF/IL-3) Interleukin 1 (IL-1) Interleukin 2 (11.-2) Interleukin 4 (IL-4) Interleukin 5 (IL-5) Interleukin 6 (IL-6) Interleukin 7 (IL-7) Interleukin 9 (11.-9) Interleukin 10 (IL-IO) Interleukin 11 (IL-11) Interleukin 12 (IL-12) Megakaryocytecolonystimulating factor (Meg~SF) Stem cell factor (SCF) Leukemia inhibitoryfactor (LIF) Oncostatin M (OSM) Macrophageinflammatory protein c((MIP-I(~)
E,Meg G,M,Eo,Meg,E G,M M,G G,M,Eo,Meg,Mast,E,Stem " T,Stem T,B B,T,G,M,Mast Eo,B B,G,Stem,Meg B,T T,Meg,Mast T Meg,B NK Meg Stem,G,E,Meg,Mast Meg ? Stem
Abbreviations: G, granulocytes;M, macrophages;Eo, eosinophils; E, erythroidcells; M, megakaryocytes; Stem, stem cells; Mast, mast cells; T, T lymphocytes;B, B lymphocytes;NK, natural killer cells.
© 1992,ElsevierSciencePublishers,(U!O
TIBS 1 7 -
AUGUST1992
painfully purified native CSFs might merely have been products of contaminant microorganisms in the tissues used as source materials. Second, the demonstration that CSFs stimulate granulocyte and macrophage formation in vivo ]° meant that the preceding 20 years of culture work had not merely been documenting some trivial, if spectacular, in vitro artifacts. Third, the demonstration that osteopetrotic (op/op) mice with severe defects in osteoclast and macrophage formation have an abnormality in their M-CSF-encoding gene that prevents transcription of M-CSF mRNA, and that the disease is correctable by the injection of M-CSFu. This provided proof that at least one CSF functions in vivo as a genuine regulator important for normal hemopoiesis.
(a)
(b)
G.cse
--°\1 7 s/~-
GM Progenitor
(c) G-CSF
G-CSF
1
~
. M-CSF ~''L-O
G-CSF
1 GM Progenitor cell
Myeloblast
1~
~roxlckD
I~'mgooytoW~
©©© Granulocytes
©© Granulocyte,*
© Macroph,,ge8
Maturo Grenulocyte
Rgure 1 Key aspects of the biology of hemopoietic regulators as exemplified by the control of granulocyte formation and function. (a) Cell production in any one lineage is dependent on regulator stimulation, but multiple regulators can directly stimulate cell proliferation because of co-expression of multiple sets of receptors on individual cells. Regulators have multiple additional actions including (b) the induction of commitment to form cells in a restricted lineage, (c) initiation of the complex events of maturation and (d) stimulation of the functional activity of the mature progeny that are finally produced.
are low (200-500 per cell) and only a small proportion of these needs to be Most of the hemopoietic regulators occupied to stimulate proliferation ~3. are glycoproteins with polypeptide Simultaneous stimulation by two or molecular masses of 14-21 kDa. Despite more relevant growth factors elicits enthis general similarity, and evidence of hanced proliferative responses. While shared functional activity, there ap- more mature precursors (usually compears to be little amino acid sequence mitted to a single lineage) can respond homology between the regulators. to stimulation by single growth factors, However, it is becoming increasingly the least mature hemopoietic cells likely that many exhibit comparable (stem cells) require combined sigthree-dimensional conformations in- nalling from multiple factors before the volving four ¢z-helical bundles, in which onset of proliferation. two helices combine to produce the The CSFs were the first regulators to reveal clearly that growth factors not active binding domain ~2. The CSFs are prototype examples of only control cell proliferation but can the mandatory role played by positive also regulate differentiation commithemopoietic regulators in controlling ment, maturation induction and the hemopoietic cell division. They can act functional activity of mature post-mitotic at concentrations of less than 1 ng ml-~ cells finally produced (Fig. 1) ~°. This to exert a concentration-dependent di- concept of polyfunctionality is now rect action in controlling cell cycling thought to apply to many, perhaps all, status, the length of the cell cycle and growth factors. With some, such as IL-6~4 the total number of progeny produced or leukemia inhibitory factor (LIF)~5, by individual precursor cells ~°. polyfunctionality also includes the The hemopoietic regulators exhibit ability to act on a wide range of nonapparent redundancy in their actions hemopoietic target cells. For example, on hemopoietic cells. For cells in any LIF also acts on osteoblasts, neurones, one lineage, there are multiple regu- hepatocytes and adipocytes. What uselators that can stimulate proliferation, ful purpose could be served in the body due to simultaneous expression of by such polyfunctional regulators? In specific receptors for the different regu- part, unwanted responses may be minilators on individual cells. Typically, mized by local production of the regureceptor numbers for any one regulator lator, resulting in effects limited to ad-
The regulators and their actions
(d)
jacent responding cells, but the question as such remains unanswered.
The receptors for hemopoietic regulators The overlapping actions of the different regulators and their polyfunctionality pose problems, since there appears to be only a single species of non-cross-reactive membrane receptor for each regulator. In the past three years an explosion of information on the receptors for hemopoietic growth factors has resolved some of these dilemmas. Some hemopoietic regulators, such as stem cell factor (SCF)]~ or M-CSF~7have classical transmembrane tyrosine kinase receptors, but the large majority of hemopoietic regulators have glycoprotein transmembrane receptors that lack a tyrosine kinase domain TM. These are now recognized to form a new group of growth factor or cytokine receptors that exhibit significant homology in their extracellular domain. Furthermore, the low-affinity tt-chains of these receptors can be converted to high-affinity receptors by association with a ~subunit. It is believed to be the ~-subunit that then initiates signalling from the ligand-receptor complex (Fig. 2). On human cells, a common ~-subunit combines competitively with the it-chains of GM-CSF, Multi-CSF or IL-5,
287
TIBS 17 - A U G U S T
which explains certain competitive interactions between the three regulators and why all ~ r e e share a common capacity to stimulate eosinophil proliferation 19. The obvious relatedness of the a-chain receptors for hemopoietic regulators indicates that they may derive from a common ancestral receptor, providing the most convincing single piece of evidence for the common evolutionary origin of many of the hemopoietic regulators. This work has now raised the further possibility that the multiple actions of individual CSFs may be mediated by multiple subunits, each initiating distinct signalling cascades that impinge on various targets within the cell.
1992
poietic regulators owing to the ability of these factors to stimulate the functional activity of mature cells. This is seen most clearly with macrophage stimulating factors such as GM-CSF. In GMCSF transgenic mice that have consistently elevated levels of GM-C~F, excess stimulation results in the production of toxic products such as tumor necrosis factor, IL-1 or plasminogen activator by the macrophages in vivo. This can lead to localized inflammatory disease and general tissue damage 23. The possible role of overproduction of hemopoietic regulators in the development of comparable disease states in humans remains to be clearly established.
0£
The future Clinical applications The hemopoietic regulators have been proven clinically to be effective in stimulating the formation of mature blood cells; erythropoietin is now the treatment of choice in the anemia of chronic renal disease 2°. This situation is comparable to the use of insulin in diabetes where the insulin-producing cells in the pancreas have been destroyed. The biology of erythropoietin production in the adult is not typical of hemopoietic regulators, in that most erythropoietin is produced in a single organ, the kidney, and erythropoietinproducing cells are damaged in renal disease. The CSFs can stimulate production of granulocytes and monocytes in disease states where production of these cells is subnormal 21,22,a situation common in cancer patients following chemotherapy or marrow transplantation. CSF production is more complex than for erythropoietin, since the CSFs originate from multiple cell types dispersed throughout the body. In healthy subjects, there are only very low levels of CSFs, but production is strongly induced by endotoxins and other microorganism products. However, these induced responses do not always result in the production of optimal levels of CSFs, even in severe aplasia or infection. This is illustrated by the ability of injected additional CSF to induce enhanced cell formation and functional activity in such patients. Most of the hemopoietic regulators now available in an active recombinant form have yet to enter clinical trials, but it is likely that at least some will be valuable in correcting hemopoietic defects, accelerating the regeneration of hemopoietic tissues after injury or in
288
Rgure 2 Schematic representation of the human membrane receptor for GM-CSF. The unique c¢-subunit contains a motif, in common with other cytokine receptors, that has spaced Cys residues (CCCC) and a Trp-Ser motif (WS). The (z-chain binds GM-CSF with low affinity, possibly in a crevice between the tandem ~barrels (stippled regions). A high-affinity receptor is then generated by crosslinking with the ~subunit, which itself contains two repeats of the cytokine receptor motif. Intracellular signalling may then be initiated by the intracytoplasmic domain of the ~subunit.
enhancing the functional activity of existing cells.
Overstimulation by hemopoietic regulators Chronic stimulation by excessive concentrations of hemopoietic regulators can lead to extreme hyperplasia in responding hemopoietic populations but is not sufficient alone to induce leukemic transformationz3-25. However, if cells acquire the abnormal ability to produce a growth factor to which they can respond it can contribute to leukemic transformation. If this is in conjunction with an intrinsic abnormality in their self-renewal during cell division, the combined abnormalities certainly lead to leukemic transformation2E Other disease states may develop as a result of excessive stimulation by the hemo-
What is the possibility that the regulatory control of hemopoiesis is typical of what occurs in other tissues? Certainly there are unusual features of hemopoiesis, such as the dispersion of cell-production sites, the complexity of specialized lineage formation from common precursor cells and the need to control mature cells in remote locations. These lend themselves to a regulatory solution that makes use of a complex of specific regulatory molecules. However, in principle, all tissues present similar regulatory problems. It is therefore reasonable to presume that the control of all tissue types is achieved by comparable regulators. The hemopoietic regulators proved relatively easy to discover because of the availability of culture systems supporting the growth and function of appropriate hemopoietic cells. Given the development of culture technology for other cell types, it is virtually certain that a very large number of specific regulators will be detectable through actions on these cells. The 20 hemopoietic regulators isolated so far are but a beginning. Major frontiers in cell biology lie waiting to be broached by the adventurous, for the golden age of regulator discovery is upon us.
References I Bradley,T. R. and Metcalf, D. (1966) Aust. J. Exp. Biol. Med. Sci. 44, 287-300 2 Ichikawa,Y., Pluznik, D. and Sachs, L. (1966) Proc. Natl Acad. Sci. USA 56, 488-495 3 Yokota,T. et al. (1984) Proc. Natl Acad. Sci. USA 81, 1070-1074 4 Fung,M-C.et al. (1984) Nature 307, 233-237 5 Gough,N. M. et al. (1984) Nature 309, 763-767 6 Souza,L. M. et al. (1986) Science 232, 61-65 7 Nagata,S. et al. (1986) Nature 319, 415-418 8 Kawasaki,E. S. et al. (1985) Science 230, 291-296 9 Jacobs, K. et al. (1985) Nature 313, 806-810
TIBS 1 7 - AUGUST1992 10 Metcalf, D. (1991) Philos. Trans. R. Soc. London Ser. B 333, 147-173 11 Wiktor-Jedrzejczak,W. et al. (1990) Proc. Natl Acad. Sci. USA 87, 4828-4832 12 Bazan, J. F. (1990) Immunol. Today 11,
350-354 13 Nicola, N. A. (1989) Annu. Rev. Biochem. 58, 45-77 14 Kishirnoto, T. (1989) Blood 74, 1-10 15 Metcalf, D. (1991) Int. J. Cell Cloning9,
DUCHENNE MUSCULAR DYSTROPHY (DMD) is a common neuromuscular disease that occurs in approximately 1 out of every 3500 live male births 3. Although the disease is present from birth and may cause developmental delays such as late onset of walking, the affected patient usually does not present until three to five years of age. Initial complaints often include leg weakness, which manifests itself in running and stair climbing difficulties and problems with rising from the floor. As the affected child grows, the loss of muscle strength causes increasing convex curvature of the spine (lordosis) and a waddle-like gait when walking. The gradual muscle-wasting generally leads to loss of ambulation by 12 years of age. The progressive loss of muscle continues throughout life with the proximal muscles affected first, followed late~" by the more distal muscles with respiratory failure and death frequently occurring by the late teens or early twenties 3. Becker muscular dystrophy (BMD) is an aUelic disorder that is less common than DMD, affecting 1 in 30 000 live male births 4. The course of BMD is much more variable and less severe than that of DMD for example, many Becker patients remain ambulatory well into adulthood and may live full and minimally restricted lives 3. At the cellular level, both DMD and BMD involve the loss of individual muscle fibers. The usual arrangement of muscle fibers is disrupted and there is marked degeneration, regeneration and fibrosis in the muscles (Fig. 1)3. While there are some abnormalities that can be detected at birth, fibrosis and fatty replacement become more obvious over time. M. D. S. Anderson and L. M. Kunkel are at
HowardHughesMedical Institute and the Division of Genetics, Children'sHospital, Harvard Medical School, Boston, MA 02115, USA.
95-108 16 Chabot, B. et al. (1988) Nature 335.88-89 17 Sherr, C. J. et al. (1985) Cell 41, 665-676 18 Bazan, J. F. (1990) Proc. Natl Acad. Sci. USA
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The molecular and biochemical basis of Duchenne muscular dystrophy
Duchenne and Becker muscular dystrophy (DMD, BMD) have both been clinically recognized for over 100 years, yet throughout much of that time nothing beyond clinical evaluation and supportive care during the disease course was available to patients. The identification of the molecular basis of DMD/BMD in 19861,2 paved the way for extensive progress toward the understanding, diagnosis and treatment of this disease.
The severity and frequency of DMD 427 kDa protein dystrophin. The sheer and BMD led to a great deal of effort in size of the gene is thought to account numerous laboratories to identify the for the unusually high mutation rate at cause of these disorders. The unequal the locus. gender distribution of the diseases gave the initial hint that the genetic location Gene characterization of DMD and BMD would be on the X The entire coding region of the gene chromosome. This was confirmed cyto- has been sequenced, providing extensive logically by analysis of rare manifesting information about the primary struccarrier females 5 and by linkage analysis 6 ture of dystrophin 8, the relationship in the early 1980s. Both types of analy- between the sizes and locations of deses narrowed the location to Xp21, letions within the gene and the clinical paving the way for identification of the presentation of the disease 9. gene itself. The increase in information about The approach used to identify the the enormous gene size has made it DMD/BMD gene set the stage for future clear that mRNA processing plays an research into other human disease- important role in DMD/BMD trancausing genes. The strategy, now scription. For example, there are a numtermed positional cloning, involved the ber of isoforms of dystrophin that differ search for and identification of a dis- at the carboxyl terminus and are proease gene with no prior knowledge of duced by alternative mRNA splicing ]°, the gene product. Based solely on the and there are at least three known map position of mutations causing the alternative transcription start points 11-]3. disease, part of the gene was identified Questions of how accurate splicing by our group in 1986 ], and a different occurs and what regulates alternative section by Ron Worton's group a few splicing of such a large gene will only be months later 2. There then followed a answered with more extensive knowlrapid description of the encoded pro- edge of the non-coding regions of the tein dystrophin 7 and the realization of DMD gene sequence. Until recently, the the complexity of the gene and its pro- examination of DMD gene processing cessing. The 2.5 million-base-pair gene has been thwarted by the enormous consists of at least 70 exons, resulting size and complexity of the DMD gene. in a 14 kb transcript which encodes the Recent construction of yeast artificial
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