© 2000 American Heart Association, Inc.
Integrative Physiology |
Leukemia Inhibitory Factor Modulates Cardiogenesis in Embryoid Bodies in Opposite Fashions
From the Institute of Biochemistry, Medical Faculty, University of Vienna, Austria.
Correspondence to Dr Georg Weitzer, Institut für Biochemie, Medizinische Fakultät, Universität Wien, Dr Bohrgasse 9/3, A-1030 Wien, Austria. E-mail gw{at}bch.univie.ac.at
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—Cardiogenesis is a multistep process regulated by a hierarchy of factors defining each developmental stage of the heart. One of these factors, leukemia inhibitory factor (LIF), a member of the interleukin-6 family of cytokines, is expressed in embryonic and neonatal cardiomyocytes and induces cardiomyocyte hypertrophy. Many aspects of embryogenesis are faithfully recapitulated during in vitro differentiation of embryonic stem cells in embryoid bodies. We exploited this model to study effects of growth factors on commitment and differentiation of cardiomyocytes and on maintenance of their phenotype. We identified LIF as a factor affecting commitment and differentiation of cardiomyocytes in an opposite manner. Diffusible LIF inhibited mesoderm formation and hampered commitment of cardiomyocytes. Lack of both the diffusible and matrix-bound isoforms of LIF in lif-/- embryoid bodies did not interfere with commitment, but it severely suppressed early differentiation of cardiomyocytes. Onset of differentiation was rescued by very low concentrations of diffusible LIF; however, consecutive differentiation was attenuated in a concentration-dependent manner by diffusible LIF both in wild-type and lif-/- cardiomyocytes. Differentiation of cardiomyocytes was severely hampered but not completely blocked in lifr-/- embryoid bodies, suggesting additional, LIF-receptor ligand independent pathways for commitment and differentiation of cardiomyocytes. At the fully differentiated state, both paracrine and autocrine LIF promoted proliferation and increased longevity of cardiomyocytes. These findings suggest that both paracrine and autocrine and both diffusible and matrix-bound isoforms of LIF contribute to the modulation of cardiogenesis in a subtle, opposite, and developmental stage–dependent manner and control proliferation and maintenance of the differentiated state of cardiomyocytes.
Key Words: leukemia inhibitory factor • cardiomyocyte development • embryonic stem cell • embryoid body
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Leukemia inhibitory factor (LIF), a member of the interleukin (IL)–6 family of cytokines, is a growth and differentiation factor with pleiotropic activities.1 LIF exists in 2 isoforms, as diffusible molecule (D-LIF) and as an extracellular matrix–bound isoform (M-LIF), with distinguishable functions.2 3 LIF binds to a receptor complex consisting of a LIF-specific receptor (LIFR) and the signal transducer gp130.4 5 This receptor complex mediates LIF signals via multiple signaling pathways following the Janus kinase/signal transducers and activators of transcription (JAK/STAT) and the mitogen-activated protein (MAP) kinase pathways.6 Several other cytokines, including IL-6, oncostatin M, IL-11, ciliary neurotrophic factor, and cardiotrophin-1 share gp130 as a common signal-transducing molecule and therefore have some similar or redundant biological activities.
LIF acts as a growth factor in hematopoiesis and on bone and neuroectodermal tissue, and it mediates immunologic responses in acute inflammation. LIF also acts as an inhibitor of differentiation on embryonic stem (ES) cells and adipocytes (for review of functions, see Reference 1 ). Notably, LIF seems to affect different developmental stages of endothelial cells, T cells, and different cells of the cardiovascular system in an opposite manner.7 8 9 10
Knockout of the LIF gene in mice demonstrated the importance of LIF for implantation of blastocysts11 and stem cell renewal.12 Overexpression of LIF in mice identified M-LIF as an inhibitor of mesodermal differentiation during gastrulation3 and demonstrated different roles for M-LIF and D-LIF in murine development. Before gastrulation, LIF is downregulated to undetectable low levels, perhaps to allow commitment to the mesodermal lineage. During embryogenesis and in many adult tissues, LIF transcripts are found, if at all, in extremely low quantities.13 Copy numbers of the transcript for M-LIF gain their highest level in the neonatal myocardium shortly before cardiomyocytes cease to divide. In primary cultures of embryonic or neonatal cardiomyocytes, LIF causes an antiapoptotic effect or hypertrophic response via the STAT3 pathway.6 14 These data support the hypothesis that LIF modulates cardiogenesis in a subtle, developmental stage–dependent, and perhaps opposite fashion. To study the function of LIF during cardiogenesis in vivo currently seems to be clearly beyond the state of the art, because D-LIF could not be detected in sera and heart tissue from healthy individuals,15 nor is it feasible to detect M-LIF at physiological concentrations. Therefore, we exploited an in vitro model of cardiogenesis to investigate the significance of LIF in cardiomyogenesis. Many aspects of cardiogenesis can be studied in differentiating aggregates of ES cells, so-called embryoid bodies.16 In embryoid bodies, all 3 germ layers and a variety of cell types form. Among them, cardiomyocytes become committed and differentiate in a manner closely recapitulating the developmental pattern of murine cardiogenesis.17
In this study, we exploit embryoid bodies to study the role of D-LIF, and indirectly of M-LIF, in commitment, differentiation, and maintenance of the phenotype of cardiomyocytes. LIF affects cardiomyogenesis in stage-dependent and opposite ways. Paracrine D-LIF negatively affects commitment and, to a lesser extent, differentiation of cardiomyocytes. In contrast, in lif-/- embryoid bodies, cardiomyocyte differentiation is severely, although not completely, blocked and can be rescued by paracrine D-LIF. Either autocrine M- and D-LIF or subpicomolar concentrations of paracrine D-LIF are necessary and sufficient for the onset of differentiation in cardiomyocytes, whereas increasing concentrations of D-LIF attenuate ongoing differentiation of cardiomyocytes. Increased inhibition of timely commitment and differentiation of cardiomyocytes in lifr-/- embryoid bodies suggests that LIFR-independent signals contribute to the development of cardiomyocytes as well. Finally, paracrine and autocrine LIFs promote proliferation and longevity of fully differentiated cardiomyocytes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Lines and Differentiation of ES Cells
The ES cell line AB2.2,18 passages 13 to 18, and SNL76/7 fibroblasts,19 passages 13 to 17, were a gift of Dr Allan Bradley (Howard Hughes Medical Institute, Houston, Tex). DIC2 lif-/- ES cells,20 DIA-RKO lifr+/-, and Hy34 lifr-/- ES cells21 were a gift of Dr Austin Smith (University of Edinburgh, Scotland). STO fibroblasts,22 passage unknown, were obtained from American Type Culture Collection. ES cells were maintained in M15 supplemented with FCS from HyClone (No. SH30070.03, lot AGL7332) on SNL76/7 feeder cells (6.1x104 feeder cells per cm2).17 23 24 For maintenance of lifr-/- ES cells, M15 was supplemented with 100 ng/mL IL-6 and 181 ng/mL soluble IL-6 receptor. Confluent ES cells maintained on SNL76/7 and STO feeder cells, respectively, were aggregated in hanging drop cultures in M15 supplemented with FCS from Sigma (No. F7524, lot 35H3379) (ratio of ES cells to feeder cells, 7.5 to 1) for 4 days and then plated at a density of 1.5 embryoid bodies per cm2.17 25 When indicated, feeder cells were separated from ES cells by absorption to culture dishes at 37°C for 45 minutes. Cardiogenesis was monitored from day 6 to day 30 after aggregation by visual inspection of beating cardiomyocytes.
To determine the effect of LIF on commitment of cardiomyocytes, ES cells maintained on SNL76/7 feeder cells were aggregated in the presence of 1, 10, 100, and 1000 pmol/L murine recombinant LIF (ESGRO, GIBCO/BRL, No. 13275-011), respectively. Embryoid bodies generated in the absence of LIF were seeded and maintained in the continuous presence of 0.01, 0.12, 1.25, 12.5, 25, and 125 pmol/L LIF starting from day 4 and day 11, respectively.
To determine the effects of feeder cells on commitment of cardiomyocytes, ES cells were maintained on STO or wild-type fibroblasts. ES cells were aggregated in the presence of increasing numbers of wild-type, lif-/-, and SNL76/7 fibroblasts, respectively. To demonstrate the influence of LIF on the differentiation of cardiomyocytes and to exclude the influence of any other factor secreted by fibroblasts, embryoid bodies were seeded in the presence of 17.6, 176, and 1760 cells/cm2 of mitotically inactivated SNL76/7, STO, wild-type, and lif-/- fibroblasts, respectively. Wild-type and lif-/- fibroblasts were isolated by serial plating of trypsinized embryoid bodies at day 14 after aggregation. To inhibit the functions of LIF, embryoid bodies were maintained in M15 containing neutralizing quantities of anti-human/mouse LIF antibody. Two different antisera (Genzyme, No. 80-2978, rabbit-derived, 10 µg/mL medium, and Oncogene Research Products, No. D05544-2, goat-derived, 0.2 µg/mL medium) were used.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Commitment of Cardiomyocytes Is Inhibited by D-LIF
D-LIF is secreted at very low rates by STO fibroblasts and at significantly increased rates by SNL76/7 fibroblasts used as feeder cells to keep ES cells in an undifferentiated state. ES cells differentiate in embryoid bodies to beating cardiomyocytes, the subject of this study. In the course of refining this in vitro model of cardiomyogenesis, to address properly the influence of autocrine and paracrine factors on commitment and differentiation of cardiomyocytes, we observed that development of cardiomyocytes in embryoid bodies (1) was inversely correlated to the concentration of D-LIF in serum supplements and (2) was significantly hampered in the presence of SNL76/7 fibroblasts. At the time when mesodermal precursors of cardiomyocytes form in embryoid bodies, presence of SNL76/7 fibroblasts significantly delayed commitment of cardiomyocytes (Figure 1A
). The number of embryoid bodies with
beating cardiomyocytes was dose-dependently reduced as
compared with embryoid bodies developed in the presence of STO
fibroblasts and in the absence of any feeder cells, respectively
(Figure 1B). Addition of LIF-neutralizing antibodies during
formation of the precardiac mesoderm significantly increased commitment
of cardiomyocytes, in the absence of fibroblasts and in the
presence of both STO and SNL76/7 fibroblasts (Figure 1C). The
effect of LIF-neutralizing antibodies was inversely correlated to the
expression level of LIF.
|
) and in the presence of
STO fibroblasts (•) and SNL76/7 fibroblasts (
), respectively. Each
data point represents mean (from 1
representative experiment of 3) of a given day±1 day
to eliminate periodic waves due to change of medium every 3 days. B,
Embryoid bodies were generated in the absence of fibroblasts (control)
and in the presence of 60 STO (STO), 60 SNL76/7 (60), and 100 SNL76/7
(100) fibroblasts per drop and 400 ES cells. C, Embryoid bodies were
generated in the absence of any fibroblasts and in the presence of 60
STO and 60 SNL cells, respectively. Half of these plates were incubated
with neutralizing anti-LIF antibodies during aggregation (+AB). B and
C, Means given in arbitrary units were calculated from data sampled
from day 6 to day 11 after aggregation, from 3 independent experiments.
Numbers of embryoid bodies checked were as follows: for B, STO, n=774;
SNL76/7, n=688; 60 SNL76/7, n=285; and 100 SNL76/7, n=1032; for C,
control/+AB, n=225; STO/+AB, n=234; and SNL/+AB, n=246. Error bars
indicate SD 
xn–1.
To demonstrate that the effects observed in the presence of SNL76/7
feeder cells were solely due to D-LIF, we aggregated ES cells in the
presence of increasing concentrations of recombinant D-LIF for 4 days.
Commitment of cardiomyocytes was attenuated and delayed by
D-LIF in a dose-dependent manner (Figure 2A). As little as 1 pmol/L D-LIF
attenuated commitment of cardiomyocytes down to 40% of the
control, and 10 pmol/L reduced the percentage of embryoid bodies with
developing cardiomyocytes to <10% (Figure 2B). As
determined by visual inspection of embryoid bodies, between 1
and 10 pmol/L D-LIF mesoderm and endoderm formed. Inhibition of
development was apparently restricted to the cardiogenic lineage,
because other cell types such as fibroblasts,
endothelial and epithelial cells, etc, developed at
rates indistinguishable from that of parallel control experiments.
|
To exclude that effects observed were not solely due to D-LIF, but to
expression of an unknown fibroblast-specific factor, we generated
embryoid bodies in the presence of SNL76/7, wild-type, and
lif-/- fibroblasts. Aggregation of ES cells in the
presence of increasing numbers of SNL76/7 fibroblasts severely hampered
commitment of cardiomyocytes in a dose-dependent manner,
whereas development of cardiomyocytes in the presence of
lif-/- fibroblasts and wild-type fibroblasts,
respectively, was not significantly altered (Figure 2C
). Weak
reduction of the number of cardiomyocytes with increasing
numbers of fibroblasts was due to exhaustion of nutrition factors in
the drop cultures. These data prove that under our experimental
conditions, cardiomyogenesis was not significantly inhibited by factors
other than D- LIF.
Cardiomyogenesis in lif-/- ES cell20
–derived embryoid bodies was hampered and reduced to 
20% of the
rate in wild-type embryoid bodies. Addition of 10 pmol/L D-LIF could
partially rescue differentiation of lif-/-
cardiomyocytes when added from day 4 to day 11 (Figure 2D). In contrast, a continuous supply of 10 pmol/L D-LIF after
day 4 could not rescue the suppressive effect exerted between days 0
and 4. Consequently, D-LIF suppresses commitment of
cardiomyocytes between days 0 and 4 in embryoid bodies, and
neither autocrine M- or D-LIF nor paracrine D-LIF is required for
proper commitment of cardiomyocytes.
Differentiation of Cardiomyocytes Is Induced and Then Attenuated
by LIF
The above findings indicated that initiation of cardiogenesis in
the primitive mesoderm takes place in the absence of LIF only. To
assess the influence of LIF on differentiation of committed
cardiomyocytes, we plated embryoid bodies at day 4 after
aggregation, first in the presence of STO, SNL76/7, wild-type, and
lif-/- fibroblasts, respectively, and second, in
the presence of D-LIF and neutralizing quantities of anti-LIF
antibodies, respectively. With proceeding differentiation, reduced
numbers of cardiomyocytes developed in the presence of
SNL76/7 fibroblasts and D-LIF, respectively (Figure 3A), whereas differentiation of
cardiomyocytes was not significantly influenced by either
STO fibroblasts or lif-/- fibroblasts (Figure 3B).
In the presence of antibodies neutralizing LIF, differentiation of
cardiomyocytes was accelerated, and percentage of embryoid
bodies developing beating foci of cardiomyocytes was
significantly increased (Figures 3A and 3B). These results
together demonstrate that subpicomolar concentrations of D-LIF
attenuate differentiation of cardiomyocytes in embryoid
bodies.
|
), and neutralizing anti-LIF antibodies (
),
respectively. B, Comparison of embryoid bodies with beating
cardiomyocytes cultivated either without any fibroblasts
(control) or on STO, lif-/-, or SNL76/7 fibroblasts
(
Differentiation but not commitment of cardiomyocytes was
severely hampered and delayed in embryoid bodies generated from
lif-/- ES cells. This phenotype could be partially
rescued by as little as 1 pmol/L D-LIF (Figure 4A). This demonstrates that autocrine
LIF, which includes M-LIF in murine cells, is essential for initiation
of differentiation of cardiomyocytes in embryoid bodies.
With increasing concentrations of D-LIF, differentiation of
cardiomyocytes in lif-/- embryoid bodies was
attenuated in a dose-dependent manner (Figure 4B). These data
demonstrate that (1) commitment of cardiomyocytes takes
place in the absence of autocrine M- and D-LIF; (2) D-LIF at very low
concentrations of <1 pmol/L is necessary and sufficient to induce
differentiation of cardiomyocytes in lif-/-
embryoid bodies; and (3) after the inductive event, D-LIF attenuates
differentiation of cardiomyocytes in a dose-dependent
manner.
|
) or lifr gene
(lifr-/-, •), or 1 copy of the lifr
gene (lifr+/-,
In the absence of LIFR in lif-/- ES
cell21 –derived embryoid bodies, cardiomyogenesis
was hampered and delayed to way below the level observed in
lif-/- embryoid bodies; however, it was not completely
blocked (Figure 4C). lifr+/- embryoid bodies
exhibited an intermediate phenotype. This result demonstrates
indirectly that (1) other IL-6 family members signaling via LIFR also
contribute to commitment and differentiation of
cardiomyocytes and (2) some cardiomyocytes
become committed and differentiate by a LIFR-independent pathway. Taken
together, differentiation of cardiomyocytes is dependent on
concentrations of <1 pmol/L of paracrine D-LIF or autocrine M-LIF and
D-LIF, and higher levels of D-LIF attenuate differentiation of
cardiomyocytes.
LIF Promotes Proliferation and Longevity of Differentiated
Cardiomyocytes
Paracrine production of LIF during in vitro
differentiation of ES cells has been shown to play a major role in stem
cell renewal,20 and nanomolar concentrations of D-LIF
exert a hypertrophic and antiapoptotic stimulus on neonatal
cardiomyocytes.6 14 Hence, we ask whether
paracrine or autocrine LIF contributes to proliferation and
maintenance of the phenotype of
cardiomyocytes in embryoid bodies. In previous experiments,
we noticed that presence of SNL76/7 fibroblasts increased longevity of
differentiated cardiomyocytes in embryoid bodies (data not
shown). To demonstrate that increased longevity of
cardiomyocytes was caused at least in part by LIF
overexpressed by SNL76/7 fibroblasts, we added increasing
concentrations of D-LIF, and LIF-neutralizing antibodies, respectively,
starting at day 11, when cardiomyocytes were fully
differentiated. D-LIF increased longevity of cardiomyocytes
by 15% (Figure 5A). In addition, a
concentration-dependent and short-lasting hypertrophic response was
observed. D-LIF acts as a proliferative stimulus on fully
differentiated cardiomyocytes in a dose-dependent manner
(Figure 5B). LIF-neutralizing antibodies blocked the
antiapoptotic effect observed in the presence of D-LIF and
SNL76/7 fibroblasts, respectively, and reduced longevity of
cardiomyocytes well below the value observed in the absence
of any fibroblasts (Figure 5C). This demonstrates that autocrine
secretion of LIF by embryoid bodies contributes to the longevity of
cardiomyocytes. In contrast to the negative effects of LIF
on commitment and differentiation of cardiomyocytes,
addition of D-LIF induces a short proliferative burst in differentiated
cardiomyocytes, and both paracrine and autocrine LIF
increase the longevity of differentiated
cardiomyocytes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In murine embryos, mesoderm emerges at the midstreak stage between 6.5 and 7.0 days postcoitum. Commitment of mesodermal cells to the cardiogenic lineage takes place during anterolateral migration at late primitive streak stage. Precardiomyocytes can be identified in the horseshoe-shaped splanchnic mesoderm 7.75 days postcoitum.26 In embryoid bodies, these developmental stages take place between 2 and 5 days after aggregation of ES cells.27 Between days 6 and 7 after aggregation, cardiomyocytes start to beat rhythmically.17 During murine embryogenesis, maternal LIF is of cardinal importance for successful implantation of blastocysts at
4.5 days
postcoitum.11 Then LIF is downregulated in the embryo
proper to facilitate gastrulation and development of mesodermal
cells,3 and transcripts are barely
detectable.13 28 Transcripts of M-LIF gain abundance after
birth, when cardiomyocytes are fully differentiated;
nevertheless, they remain in the cell cycle for another dozen days. In
embryoid bodies, downregulation of LIF during gastrulation was mimicked
by aggregating ES cells in medium supplemented with serum containing
very low levels of LIF. By this measure, we were able to recapitulate
many aspects of cardiomyogenesis in vitro and to address the opposite
effects of LIF during commitment, differentiation, and
maintenance of cardiomyocytes in embryoid
bodies. In the presence of D-LIF from the very beginning of ES cell differentiation, we observed a dose-dependent inhibition of mesoderm formation similar to effects observed with P19 embryonic carcinoma cells.29 This suggests that D-LIF has to be downregulated before gastrulation to levels well below 1 pmol/L to allow mesoderm to form and to progress along the cardiogenic lineage. D-LIF at 1 nmol/L inhibited pathways leading to endodermal cells distal to the Reichert’s membrane so that embryoid bodies no longer were able to adhere to the culture dishes. To test whether formation of mesoderm per se was inhibited or only consecutive differentiation of mesodermal cells along the cardiogenic lineage was attenuated in embryoid bodies, we added D-LIF after mesodermal precursors had become committed to the cardiogenic lineage after day 4. Any concentration tested starting from as little as 10 fmol/L LIF attenuated differentiation of cardiomyocytes. Released block of differentiation by neutralizing anti-LIF antibodies corroborated the negative effect of D-LIF on cardiomyocyte development. However, in the complete absence of LIF in lif-/- embryoid bodies, timely differentiation of cardiomyocytes was hampered and delayed. This suggests, first, that at least low levels of autocrine D- and/or M-LIF are necessary for differentiation of cardiomyocytes, and second, that LIF mediates a short-lasting inductive effect triggering cardiomyocyte differentiation. Most recently, a similar role has been demonstrated for LIF in the mesenchymal-to-epithelial conversion in the metanephron.30 The dominant-negative effect of LIF on commitment and differentiation may contribute to the proper development of the embryonic heart and to the modulation of pathophysiological processes in myocardial disease.
In lif-/- embryoid bodies, lack of autocrine M-and D-LIF hampered and delayed cardiomyogenesis significantly; however, at times when wild-type cardiomyocytes usually ceased to divide, a second population of cardiomyocytes stepped into existence. From this we may speculate that at least 2 populations of cardiomyocytes form in embryoid bodies. We observed a LIF-sensitive lineage of cardiomyocytes concomitantly developing at the time when early heart development takes place in the murine embryo and a second, perhaps LIF-independent, lineage developing around day 20 after ES cell aggregation. The later one was observed in lif-/- and lifr-/- embryoid bodies and in wild-type embryoid bodies, in which development of the first lineage was blocked by paracrine D-LIF (A. Bader and G. Weitzer, unpublished results, 1999). Cardiomyocytes formed later on during development may be triggered to differentiate by a different signal molecule in the absence of autocrine LIF. A good candidate in lif-/- embryoid bodies is cardiotrophin-1, which originally has been isolated from embryoid bodies.31 A second LIF-independent pathway triggering cardiomyogenesis is suggested by the incomplete block of commitment and differentiation of cardiomyocytes in lifr-/- embryoid bodies. This late onset of cardiomyocyte development may be caused by a factor secreted from embryoid bodies at later stages of development, which obviously does not belong to the LIFR ligand family. A candidate factor may be the "ES cell renewal factor" isolated from lif-/- endodermal cells.20
Once cardiomyocytes were fully developed, D-LIF exerted an opposite function, as observed during the development of cardiomyocytes. With increasing concentrations of LIF, longevity of cardiomyocytes was significantly increased by suppressing apoptosis, as has been demonstrated previously for primary cultures of cardiomyocytes.14 In addition, a weak and short-lasting hypertrophic response in cardiomyocytes was observed. We think that LIF may delay the irreversible exit of fully differentiated cardiomyocytes from the cell cycle in the neonatal heart, because in vivo M-LIF is expressed concomitantly in the murine myocardium.13
The physiological relevance of the dominant-negative effect of LIF during cardiogenic commitment and differentiation may be illustrated by the fact that this process in embryoid bodies correlates with inhibition of mesoderm formation in embryos overexpressing M-LIF.3 The temporally increased proliferation and longevity of fully differentiated cardiomyocytes in embryoid bodies correlate with the regain of detectable levels of M-LIF transcripts in the neonatal heart.13 Opposite functions of LIF, as demonstrated for cardiomyocytes, have also been reported for various phases of T-cell development,9 suggesting that paracrine and autocrine LIF-mediated signal transduction may switch from positive to negative regulation, and vice versa, depending on developmental and physiological stages of a particular cell type. LIF may not only share functional redundancy with other IL-6 family members because of the shared usage of LIFR and gp130, but for the same reason, and provided that different binding specificity exists, LIF may act as a competitive inhibitor of factors such as cardiotrophin-1.32 This may explain how LIF can act both as an activator and an inhibitor with subtle regulatory roles in developing cardiomyocytes. The mutual roles of LIF may be additionally tuned by LIFR, expressed as soluble and high-affinity, membrane-bound, and gp130-associated isoforms.33 Altered ligand binding affinity and different expression patterns of these receptors may modulate the functions of LIF or the susceptibility of cells to LIF as well. The putative roles of these LIFR isoforms in cardiomyocytes remain to be studied.
LIF seems to be dispensable for cardiogenesis in mice11 ; however, we were able to demonstrate that subpicomolar concentrations of LIF modulate the cardiogenic pathway in a subtle and disparate fashion, and that a LIF-null allele severely hampers cardiomyogenesis in embryoid bodies. In knockout mice, LIF is most likely substituted by other members of the IL-6 family, because knockout of gp130 indeed led to embryonic lethal defects in the ventricular myocardium,34 and knockout of the lifr gene led to pleiotropic effects causing perinatal death.35 This suggests that LIF contributes, together with a plethora of other factors, to the proper formation and maintenance of the vertebrate heart.
Much is currently known regarding the development of the heart,36 but little is understood regarding the maintenance of the differentiated state of cardiomyocytes. Several lines of evidence suggest that differentiation requires continuous regulation by transcription factors and growth factors. A hierarchy of regulators leads to and defines each developmental stage of cardiogenesis. Once cardiomyocytes are fully differentiated, they have to perform their vitally important function throughout the life of an organism. To maintain that stable state, feedback mechanisms may be required to circumvent the regulatory hierarchy and sustain a threshold level of critical regulators of the cardiac phenotype. In the case of myoblasts, such a mechanism may be provided by the myoD family of myogenic transcription factors and by transcription of the 3'-untranslated regions of structural genes.37 A role in maintenance of the cardiac phenotype has been demonstrated for the muscle-specific intermediate filament protein desmin38 39 ; however, little else is known about regulators of the fully differentiated state of cardiomyocytes. We suggest that M- and D-LIF exert such a maintaining role at very low concentrations, presumably together with a plethora of other factors, owing to the shared usage of gp130. The presence of very low levels of both M- and D- LIF in the myocardium seems to modulate cardiogenesis in a subtle, time-sensitive, and opposite fashion and to control proliferation and maintenance of the fully differentiated state of cardiomyocytes.
|
Acknowledgments |
|---|
This study was supported by the Jubiläumsfonds der Österreich-ische Nationalbank (No. 5669) and the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (P11189-MED and P12206-GEN, to G.W.). We are grateful to Dr Allan Bradley for the AB2.2 ES cells and the SNL76/7 fibroblasts and Dr Austin Smith for lif-/-, lifr+/-, and lifr-/- ES cells. We also thank Karin Habegger and Sabine Enzinger for their excellent technical assistance and our colleagues Alexandra Höllrigl and Sonja Puz, who helped in performing experiments for the timely improvement of this manuscript.
Received August 10, 1999; accepted December 27, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- Taupin JL, Pitard V, Dechanet J, Miossec V, Gualde N, Moreau JF. Leukemia inhibitory factor: part of a large ingathering family. Int Rev Immunol. 1998;16:397–426.[Medline] [Order article via Infotrieve]
- Rathjen PD, Toth S, Willis A, Heath JK, Smith AG. Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage. Cell. 1990;62:1105–1114.[Medline] [Order article via Infotrieve]
-
Conquet F, Peyriéras N, Tiret L, Brûlet P.
Inhibited gastrulation in mouse embryos overexpressing the leukemia
inhibitory factor. Proc Natl Acad Sci U S A. 1992;89:8195–8199.
[Abstract/Free Full Text] -
Godard A, Heymann D, Raher S, Anegon I, Peyrat MA, Le
Mauff B, Mouray E, Gregoire M, Virdee K, Soulillou JP, Moreau JF,
Jaques Y. High and low affinity receptors for human interleukin for DA
cells/leukemia inhibitory factor on human cells: molecular
characterization and cellular distribution. J Biol
Chem. 1992;267:3214–3222.
[Abstract/Free Full Text] -
Gearing DP, Comeau MR, Friend DJ, Gimpel SD, Thut CJ,
McGourty J, Brasher KK, King JA, Gillis S, Mosley B, Ziegler SF, Cosman
D. The IL-6 signal transducer, gp130: an oncostatin M receptor and
affinity converter for the LIF receptor. Science. 1992;255:1434–1437.
[Abstract/Free Full Text] -
Kunisada K, Tone E, Fujio Y, Matsui H,
Yamauchi-Takihara K, Kishimoto T. Activation of gp130 transduces
hypertrophic signals via STAT3 in cardiac myocytes.
Circulation. 1998;98:346–352.
[Abstract/Free Full Text] - Paradis H, Arceci RJ, Adams LC, Gendron RL. Differentiation responses of embryonic endothelium to leukemia inhibitory factor. Exp Cell Res. 1998;240:7–15.[Medline] [Order article via Infotrieve]
- Pepper MS, Ferrara N, Orci L, Montesano R. Leukemia inhibitory factor (LIF) inhibits angiogenesis in vitro. J Cell Sci. 1995;108:73–83.[Abstract]
- Towle MF, Mondragon-Escorpizo M, Norin A, Fukada K. Deprivation of leukemia inhibitory factor by its function-blocking antibodies augments T cell activation. J Interferon Cytokine Res. 1998;18:387–392.[Medline] [Order article via Infotrieve]
- Kirby ML, Kumiski DH, Myers T, Cerjan C, Mishima N. Backtransplantation of chick cardiac neural crest cells cultured in LIF rescues heart development. Dev Dyn. 1993;198:296–311.[Medline] [Order article via Infotrieve]
- Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, Abbondanzo SJ. Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature. 1992;359:76–79.[Medline] [Order article via Infotrieve]
- Escary JL, Perreau J, Dumenil D, Ezine S, Brulet P. Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation. Nature. 1993;363:361–364.[Medline] [Order article via Infotrieve]
- Robertson M, Chambers I, Rathjen P, Nichols J, Smith A. Expression of alternative forms of differentiation inhibiting activity (DIA/LIF) during murine embryogenesis and in neonatal and adult tissues. Dev Genet. 1993;14:165–173.[Medline] [Order article via Infotrieve]
- Sheng Z, Pennica D, Wood WI, Chien KR. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development. 1996;122:419–428.[Abstract]
- Waring P, Wycherley K, Cary D, Nicola N, Metcalf D. Leukemia inhibitory factor levels are elevated in septic shock and various inflammatory body fluids. J Clin Invest. 1992;90:2031–2037.
-
Hescheler J, Fleischmann BK, Lentini S, Maltsev VA,
Rohwedel J, Wobus AM, Addicks K. Embryonic stem cells: a model to study
structural and functional properties in cardiomyogenesis.
Cardiovasc Res. 1997;36:149–162.
[Free Full Text] - Weitzer G, Milner DJ, Kim JU, Bradley A, Capetanaki Y. Cytoskeletal control of myogenesis: a desmin null mutation blocks the myogenic pathway during embryonic stem cell differentiation. Dev Biol. 1995;172:422–439.[Medline] [Order article via Infotrieve]
- Soriano P, Montgomery C, Geske R, Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991;64:693–702.[Medline] [Order article via Infotrieve]
- McMahon AP, Bradley A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell. 1990;62:1073–1085.[Medline] [Order article via Infotrieve]
- Dani C, Chambers I, Johnstone S, Robertson M, Ebrahimi B, Saito M, Taga T, Li M, Burdon T, Nichols J, Smith A. Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway. Dev Biol. 1998;203:149–162.[Medline] [Order article via Infotrieve]
-
Aubert J, Dessolin S, Belmonte N, Li M, McKenzie FR,
Staccini L, Villageois P, Barhanin B, Vernallis A, Smith AG, Ailhaud G,
Dani C. Leukemia inhibitory factor and its receptor promote
adipocyte differentiation via the mitogen-activated protein
kinase cascade. J Biol Chem. 1999;274:24965–24972.
[Abstract/Free Full Text] -
Martin GR, Evans MJ. Differentiation of clonal lines of
teratocarcinoma cells: formation of embryoid bodies in vitro.
Proc Natl Acad Sci U S A. 1975;72:1441–1445.
[Abstract/Free Full Text] - Yoshida K, Chambers I, Nichols J, Smith A, Saito M, Yasukawa K, Shoyab M, Taga T, Kishimoto T. Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gp130 signalling pathways. Mech Dev. 1994;45:163–171.[Medline] [Order article via Infotrieve]
- Nichols J, Chambers I, Smith A. Derivation of germline competent embryonic stem cells with a combination of interleukin-6 and soluble interleukin-6 receptor. Exp Cell Res. 1994;215:237–239.[Medline] [Order article via Infotrieve]
- Maltsev VA, Rohwedel J, Hescheler J, Wobus AM. Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types. Mech Dev. 1993;44:41–50.[Medline] [Order article via Infotrieve]
- Kuisk IR, Li H, Tran D, Capetanaki Y. A single MEF2 site governs desmin transcription in both heart and skeletal muscle during mouse embryogenesis. Dev Biol. 1996;174:1–13.[Medline] [Order article via Infotrieve]
- Leahy A, Xiong JW, Kuhnert F, Stuhlmann H. Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. J Exp Zool. 1999;284:67–81.[Medline] [Order article via Infotrieve]
- Nichols J, Davidson D, Taga T, Yoshida K, Chambers I, Smith A. Complementary tissue-specific expression of LIF and LIF-receptor mRNAs in early mouse embryogenesis. Mech Dev. 1996;57:123–131.[Medline] [Order article via Infotrieve]
- Pruitt SC, Natoli TA. Inhibition of differentiation by leukemia inhibitory factor distinguishes two induction pathways in P19 embryonal carcinoma cells. Differentiation. 1992;50:57–65.[Medline] [Order article via Infotrieve]
- Barasch J, Yang J, Ware CB, Taga T, Yoshida K, Erdjument-Bromage H, Tempst P, Parravicini E, Malach S, Aranoff T, Oliver JA. Mesenchymal to epithelial conversion in rat metanephros is induced by LIF. Cell. 1999;99:377–386.[Medline] [Order article via Infotrieve]
-
Pennica D, King KL, Shaw KJ, Luis E, Rullamas J, Luoh
SM, Darbonne WC, Knutzon DS, Yen R, Chien KR, Baker JB, Wood WI.
Expression cloning of cardiotrophin 1, a cytokine that induces
cardiac myocyte hypertrophy. Proc Natl Acad Sci
U S A. 1995;92:1142–1146.
[Abstract/Free Full Text] - Pennica D, Wood WI, Chien KR. Cardiotrophin-1: a multifunctional cytokine that signals via LIF receptor-gp 130 dependent pathways. Cytokine Growth Factor Rev. 1996;7:81–91.[Medline] [Order article via Infotrieve]
- Chambers I, Cozens A, Broadbent J, Robertson M, Lee M, Li M, Smith A. Structure of the mouse leukaemia inhibitory factor receptor gene: regulated expression of mRNA encoding a soluble receptor isoform from an alternative 5' untranslated region. Biochem J. 1997;328:879–888.
-
Yoshida K, Taga T, Saito M, Suematsu S, Kumanogoh A,
Tanaka T, Fujiwara H, Hirata M, Yamagami T, Nakahata T, Hirabayashi T,
Yoneda Y, Tanaka K, Wang WZ, Mori C, Shiota K, Yoshida N, Kishimoto T.
Targeted disruption of gp130, a common signal transducer for the
interleukin 6 family of cytokines, leads to myocardial and
hematological disorders. Proc Natl Acad Sci U S A. 1996;93:407–411.
[Abstract/Free Full Text] - Ware CB, Horowitz MC, Renshaw BR, Hunt JS, Liggitt D, Koblar SA, Gliniak BC, McKenna HJ, Papayannopoulou T, Thoma B, Cheng L, Donavan PJ, Peschon JJ, Bartlett PF, Willis CR, Wright BD, Carpenter MK, Davison BL, Gearing DP. Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death. Development. 1995;121:1283–1299.[Abstract]
- Fishman MC, Olson EN. Parsing the heart: genetic modules for organ assembly. Cell. 1997;91:153–156.[Medline] [Order article via Infotrieve]
- Rastinejad F, Blau HM. Genetic complementation reveals a novel regulatory role for 3' untranslated regions in growth and differentiation. Cell. 1993;72:903–917.[Medline] [Order article via Infotrieve]
-
Milner DJ, Weitzer G, Tran D, Bradley A, Capetanaki Y.
Disruption of muscle architecture and myocardial degeneration in mice
lacking desmin. J Cell Biol. 1996;134:1255–1270.
[Abstract/Free Full Text] - Li Z, Colucci-Guyon E, Pinçon-Raymond M, Mericskay M, Pournin S, Paulin D, Babinet C. Cardiovascular lesions and skeletal myopathy in mice lacking desmin. Dev Biol. 1996;175:362–366.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  R. E. Davey, K. Onishi, A. Mahdavi, and P. W. Zandstra LIF-mediated control of embryonic stem cell self-renewal emerges due to an autoregulatory loop FASEB J, July 1, 2007; 21(9): 2020 - 2032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Wobus and K. R. Boheler Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy Physiol Rev, April 1, 2005; 85(2): 635 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Berry, T. J. Pirolli, V. Jayasankar, K. J. Morine, M. A. Moise, O. Fisher, T. J. Gardner, P. H. Patterson, and Y. J. Woo Targeted overexpression of leukemia inhibitory factor to preserve myocardium in a rat model of postinfarction heart failure J. Thorac. Cardiovasc. Surg., December 1, 2004; 128(6): 866 - 875. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Goldenthal and J. Marin-Garcia Stem cells and cardiac disorders: an appraisal Cardiovasc Res, May 1, 2003; 58(2): 369 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Gepstein Derivation and Potential Applications of Human Embryonic Stem Cells Circ. Res., November 15, 2002; 91(10): 866 - 876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. Boheler, J. Czyz, D. Tweedie, H.-T. Yang, S. V. Anisimov, and A. M. Wobus Differentiation of Pluripotent Embryonic Stem Cells Into Cardiomyocytes Circ. Res., August 9, 2002; 91(3): 189 - 201. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||