Download Hedgehog proteins: expression and function in the thymus

Revisión
Inmunología
Vol. 22 / Núm 1/ Enero-Marzo 2003: 17-26
Hedgehog proteins:
expression and function in the thymus
R. Sacedón1, A. Varas2, C. Hernández-López2, M.C. Gutiérrez-de Frías2,
S.V. Outram3, T. Crompton3, A. Vicente1, A. Zapata2
1Department of Cell Biology, Faculty of Medicine, Complutense University.
Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain.
3Department of Biology, Imperial College of Science, Technology, and Medicine, London.
2
PROTEÍNAS HEDGEHOG: EXPRESIÓN Y FUNCIÓN EN EL TIMO
RESUMEN
Evidencias crecientes demuestran que los morfógenos clásicos, altamente conservados durante la evolución y tradicionalmente implicados en el desarrollo embrionario, donde determinan diferenciación celular y patrones de desarrollo, son expresados en tejidos adultos, como son la médula ósea y el sistema inmunitario. Estas moléculas podrían ser piezas importantes del rompecabezas que orquesta la diferenciación y la homeostasis del sistema inmune, aunque sea sólo recientemente cuando estamos
comenzando a entender donde encajan en el entramado del sistema inmune. En esta revisión, describimos la ruta de señalización de las proteínas Hedgehog (Hh) centrándonos en su implicación en la diferenciación de las células T y su posible conexión
con otros morfógenos, como son las proteínas morfogénicas del
hueso (Bone Morphogenetic Proteins, BMPs)
ABSTRACT
Growing evidence demonstrates that classical ancient morphogens traditionally implicated in embryonic processes, determining cell
fate and developmental patterns, are expressed in adult tissues including bone marrow and immune system. They could represent important
pieces of the puzzle that orchestrate immune system differentiation and
homeostasis, although only recently we are beginning to understand
where they fit in the immune system millieu. In this review we outline
the signalling cascade of the Hedgehog (Hh) proteins, focusing on its
involvement in T-cell differentiation and the possible connections with
other morphogens such as Bone Morphogenetic Proteins (BMPs).
KEY WORDS: Hedgehog (Hh) proteins / Bone Morphogenetic Proteins
(BMPs) / T differentiation / Thymus.
PALABRAS CLAVE: Proteínas Hedgehog (Hh) / Proteínas morfogénicas del hueso / Diferenciación T / Timo.
THE ANCIENT HEDGEHOG FAMILY
Hh genes encode a family of secreted proteins the activities
of which include those of classic morphogens, orchestrating
embryonic development, but also the regulation of cell survival
and proliferation. Knowledge of their pleiotropic functions
is profoundly complex as they elicit different or even opposed
effects depending on the dose and/or target cell.
Initially described as a segment polarity gene in Drosophila,
hh proteins were named on the basis of the appearance of
mutant larvae. Disruption of hh gene leads to Drosophila
larvae covered by a continuos lawn of denticles projecting
from their cuticle as spines of a hedgehog, which suggested
to Nüsslein-Volhard and Wieschands(1) the name of this
family of proteins.
17
HEDGEHOG PROTEINS: EXPRESSION AND FUNCTION IN THE THYMUS
Sonic Hh (Shh), named after Sega hedgehog hero, and
Indian and Desert Hh (Ihh and Dhh, respectively), both
referred as two hedgehog species, were later identified in
vertebrates(2), from fish to mammals. They are key players
in most of vertebrate developmental processes and so,
implicated in several types of human congenital abnormalities,
including holoprosoencephalia, and cancer(3-5).
The most pleiotropic of vertebrate Hh molecules is Shh.
It orchestrates diverse events such as left-right asymmetry(6–8),
dorso-ventral patterning of the central nervous system(2,9–12)
and somites(13,14), as well as limb morphogenesis(15,16). Shh
is also involved in the development of lung(17), tooth(18,19),
hair(20), pancreas(21,22) and tongue(23). Haematopoietic stem
cells are Shh targets (24) and, as discussed later, T cell
differentiation(25) and proliferation(26) seem to be also modulated
by this Hh protein.
On the other hand, Ihh plays key functions in the
development of bone and cartilage(27) as well as pancreatic
morphogenesis(22). In addition, it activates haematopoiesis
and vasculogenesis during early post-implantation
development(28). Its involvement in development of the
mammary gland(29) as well as in differentiation of visceral
endoderm(30) has been also suggested.
Dhh function seems to be more restricted. Its implication
in the development of testicles and external genitalia(31-33)
and in the formation of peripheral nerve ensheathment(33,34)
has been demonstrated.
All the described developmental processes are regulated
by Hh molecules not only because of their capacity to
determine cell fate but also by their ability to modulate cell
survival and proliferation. The first evidence supporting a
role for Hh in cell proliferation raises from the evidence
that mutations that activate the Hh cascade lead to several
types of cancer such as basal cell carcinoma, medulloblastoma,
rhabdomyosarcoma(3-5). Nevertheless, the molecular basis
that link Hh signalling to cell cycle progression remains
unclear and only a few recent studies have approached this
question. For example, in cerebellar granule neurone
precursors, Shh promotes cell division regulating levels of
proteins implicated in G1-S restriction point, including
cyclins D1, D2 and E(35). Similar results are observed in
Drosophila eye development where Hh induces the expression
of cyclins D and E(36). Furthermore, the interaction of the
Hh receptor with the phosphorylated form of cyclin B, and
thereby its cytoplasmic retention, has also related the Hh
cascade with the regulation of G2-M cell cycle progression,
although its physiological significance is not yet understood(37).
Not only a tightly controlled cell proliferation but also
programmed cell death are essential to proper organogenesis.
On this regard, Shh elicits apoptotic or antiapoptotic effects
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VOL. 22 NUM. 1/ 2003
in different organs. Shh survival actions in somites(38),
neurons(39), neural crest cells(40) and muscle cells of the
limb(41,42) contrast with its apoptotic effect in posterior limb
bud cells(43). Again, further investigations are required to
a better knowledge of the molecular basis of these dual
roles.
HEDGEHOG SIGNALLING CASCADE
Hh proteins are synthesised in a precursor form that
is autocatalitically processed rendering an active N-terminal
fragment that is secreted(44). Concomitantly to this posttranslational cleavage, the signalling molecules are covalently
coupled to cholesterol at their COOH-terminal ends, which
is necessary for their movement across the target field(45,46).
A second lipid modification, palmitoylation, occurs in the
NH2-terminal end of a part of the proteins increasing their
signalling activity in vitro(47) and in vivo(48). Both modifications
participate in tethering the proteins to the cell membrane
in synthesising cells, although they are neither necessary
for receptor recognition nor affect receptor binding affinity.
On the other hand, the Drosophila model provides evidence
that Hh target expression requires the active release of lipid
modified Hh from the surface membrane of the secreting
cell. This is mediated by a membrane molecule called
Dispatched(49,50).
All Hh proteins share an apparently exclusive signalling
pathway (Fig. 1) and differences among the particular roles
of Shh, Ihh and Dhh during vertebrate development are
really provided, and delimited by their distinct temporal
and spatial expression domains (31). Pathi et al. (51), also
demonstrated that all three proteins can elicit similar biological
responses, but that their relative potencies differ in an assaydependent manner. In most cases, data revealed a stronger
activity of Shh followed by Ihh and Dhh, with the lowest
potency. However, these quantitative differences are not
based on their ability to bind to the receptor. Additional
components of the Hh cascade, which modulate the responses
of the three Hh proteins as well as the requirement of different
range of required signalling potencies for each particular
biological event, could explain the existence of three different
Hh proteins in vertebrates.
Patched (Ptc), a politopic transmembrane protein
with homology to transporter or channel proteins(52-54), and
Smoothened (Smo), a serpentine transmembrane protein
reminiscent of G-protein coupled receptors(55,56), represent
the membrane components of the Hh cascade (Fig. 1). Ptc
is the ligand binder and Smo transduces the intracellular
signal. In the absence of ligand, Ptc exerts an inhibitory
effect on Smo activity that is abrogated after Hh binding.
INMUNOLOGÍA
R. SACEDÓN ET AL.
Figure 1. Hh Signalling cascade in vertebrates. Hh proteins (Shh, Ihh, Dhh) are synthesised and secreted but they can also attach to the cell membrane through
cholesterol or palmitoylation modification. Target field establishment is promoted by cell to cell transfer and through the building of multimers, possibly mediated
by a homologue of the Drosophila membrane protein, Dispatched. Heparan sulfate associated to proteoglycans, such as syndecans, and/or other extracellular
matrix proteins modulate or promote Hh binding to its receptor. Patched represents the Hh ligand binder while Smoothened initiates the Hh signalling cascade
that activates transcriptional activities of Gli proteins. PKA inhibits Smoothened function. Additionally, other cell membrane proteins such as Hip, Megalin
and Gas1 modulate Hh levels or even could initiate new intracellular cascades (see text for more details).
In the absence of Ptc, Smo exhibits a tonic activity(55,57,58).
The mechanism underlying Ptc inhibitory function is under
debate, although recent evidence reinforces the idea that
the permanent physical interaction between Ptc and Smo
is not necessary. On the contrary, this inhibition would
involve the covalent modification of Smo, combined with
the regulation of the vesicular trafficking(59-63). Direct protein
kinase A (PKA) phosphorylation of Smo in its COOHterminal domain has been also proposed to inhibit Hh
cascade targeting Smo to degradation (Fig. 1)(59).
The intracellular Hh transduction pathway has been
also more extensively studied in Drosophila. Smo derepression
leads to gene expression regulation, modifying the activity
of a transcription factor, Cubitus interruptus (Ci)(64). In
the fly, in the absence of Hh, Ci is retained in the cytosol
forming a complex with several segment polarity proteins,
the kinase fused, costal- 2 and suppresser of fused (SuFu).
These proteins mediate and regulate Ci anchorage to
cytoskeletal microtubules and, therefore, cytoplasmicnuclear shuttling(65-69). Ci is phosphorylated by PKA and
other kinases. PKA phosphorylation of Ci is required for
Ci to be cleaved yielding a transcriptional repressor. In fact,
PKA mutant cells show constitutive activation of Hh target
genes(70). Hh signalling leads to phosphorylation of Fused
and dephosphorylation of Ci. The change in phosphorylation
pattern leads to the inhibition of repressor formation. Then,
Ci is translocated to the nucleus where, alone or in collaboration
with CREB Binding Protein or SuFu, it interacts with DNA
and activates Hh target genes(71).
In vertebrates three gli genes (gli1, gli2, gli3) are the Ci
homologous (Fig. 1). Nevertheless, the functions of the three
Gli transcription factors are not redundant, since they exhibit
different expression patterns during development and their
cellular activities are significantly distinct(72).
Gli1 is a Hh-dependent activator of Hh target genes
whereas Gli 3 is mainly a Hh-dependent repressor. PKA
activity also promotes Gli 3 repression by inducing its
cleavage, which yields a repressor form, as described for
Ci(72,73). All three Gli proteins are Hh target genes but whereas
gli-1 expression is induced by Hh, the expression of gli-3 is
repressed(74,75). On the contrary, Gli2 can function as repressor
or activator of Hh target genes: it acts as activator in a
Hh-dependent manner or as repressor independently of
Hh(76-79).
Not all Gli functions are associated with the Hh cascade.
Gli 2 and Gli3 are expressed in regions, such as the dorsal
neural tube, that require absence of Hh signalling(75,80-82).
This may correlate with their Hh-independent inhibitory
activities. Moreover, Hh signalling cascade is not restricted
to Gli function(83). There is a dramatic in vitro impairment
of neural crest cell migration induced by Shh that is insensitive
both to inhibition of protein synthesis or to cyclopamine
treatment, a plant steroid alkaloid that blocks Smo function(60,84).
On this basis, Testaz et al.(83) demonstrated a biological Shh
19
HEDGEHOG PROTEINS: EXPRESSION AND FUNCTION IN THE THYMUS
action independent of the classic Ptc-Smo-Gli signalling
pathway, although they did not propose any alternative.
MORE THAN A SECRETED PROTEIN THAT BINDS
TO ITS RECEPTOR
Proper Hh function requires a precise regulation in time
and/or space of its signalling. This is achieved in vivo by
a fairly complex system that would work by different
mechanisms: a) determining the levels of available Hh
molecules in a target field not only regulating their secretion
and diffusion, but also their cell membrane sequestration
and intracellular degradation; b) modulating the cell
responsiveness at receptor and transducing levels.
As any morphogen, Hh proteins are able to influence
cells located in short or long distances from secreting cells.
The appearance of a gradient may be originated by passive
diffusion and/or facilitated transport mediated by a cell to
cell membrane transfer or even a transcytosis mechanism,
although this last issue remains obscure(85,86). It is known
that cholesterol tethering to the membrane is required for
the establishment of Shh long range field of action(46). In a
recent work, Zhen et al.(86) demonstrated the existence of
a freely diffusive multimeric form of cholesterol modified
Shh. This Hh multimer hides the lipid attachments in its
interior, increasing its diffusion field. The authors proposed
that concentration in lipid rafts of Hh cholesterol modified
molecules and the implication of a possible vertebrate
homologue of Dispatched, could lead to the building of
such a multimer (Fig. 1). On the other hand, Hh binding to
Ptc has a dual role limiting Hh diffusion and reducing its
levels by endocytosis, although a transduction role of such
endocytosis could not be discarded (Fig. 1). Thus, the increase
of Ptc levels after Hh signalling represents a negative feedback
loop(87-89).
McCarthy et al.(90) have proposed the existence of a new
molecule that may participate in Hh function, an endocytic
receptor belonging to the low density lipoprotein receptor
family, megalin. This molecule mediates ligand endocytosis
in the apical surface of different epithelia, and subsequently,
their lysosomal degradation or, alternatively, their transcytosis.
However, it is not possible to discard that megalin can also
initiate a signalling cascade after Shh binding (Fig. 1).
Another protein that also binds Hh molecules is the Hhinteracting protein (Hip). Hip is a glycosilated membrane
protein that is expressed next to cells that produce Hh, and
binds Hh proteins with the same affinity than Ptc. No
signalling cascade has been demonstrated after Hip-Hh
interaction, indicating that the main function of Hip would
be to attenuate Hh activities(91) (Fig. 1).
20
VOL. 22 NUM. 1/ 2003
Recently, Gas 1 has been demonstrated to bind Shh and
block the growth-stimulating activity of moderate doses
of Shh(92). In cultured cells, Gas 1 exerts a p53-dependent
growth cell arrest apparently in the absence of any known
ligand(93), although in other systems this molecule does not
cause cell proliferation blockage(93). This evidence suggests
the existence of a Shh-independent role of Gas 1, which
may be more than a Shh sequester; after Shh binding, Gas1
could originate a new Ptc/Smo-independent intracellular
cascade (Fig. 1).
Modulation of cell responsiveness is also crucial to
proper Hh function. As described in other signalling
pathways, positive and negative feedback loops are
established. Ptc and Gli1 levels increase in response to Hh.
On the contrary, Gli3 and Shh levels are inhibited. Membrane
Smo levels have been also demonstrated to increase after
Hh signalling. However, it is not clear whether this is merely
an effect of Ptc derepression without a real increase in Smo
transcription(59-63).
In addition, two extracellular matrix glycoproteins,
laminin and vitronectin, have been demonstrated to modulate
proliferative and differentiation responses to Shh (94,95).
Disruption of heparan sulphate proteoglycans (HSPG)
synthesising enzyme Tout-velu in Drosophila suggests a role
for these extracellular matrix molecules in long range Hh
activities (96,97). Rubin et al. (98) extended these results to
vertebrates and demonstrated that Shh-HSPG binding
significantly increases proliferative responses to Hh without
affecting Ptc binding affinity (Fig. 1).
HH SIGNALLING REGULATES T CELL
DIFFERENTIATION
Cytokine and pre-TCR are considered essential signalling
pathways for early T cell maturation. We have recently
reported the involvement of Hh molecules in T cell
development, demonstrating that Shh plays a key role
regulating early T cell proliferation and differentiation(25).
Shh is the main Hh molecule secreted in the fetal and
adult mouse thymus, and is produced by the thymic
epithelium. Ihh expression is mainly associated to the blood
vessels from the medullary region in the adult thymus, and
Dhh is not detected in the organ(25).
Further studies demonstrate that mRNA of both
components of the Hh receptor, Ptc and Smo, are also present
in fetal and adult thymocytes. The flow cytometric analysis
of Smo expression shows that most of CD4–CD8– double
negative (DN) thymocytes express high levels of the signalling
component of the Hh receptor, whereas cell surface expression
of Smo is downregulated in the CD4+CD8+ double positive
R. SACEDÓN ET AL.
INMUNOLOGÍA
a
b
Percentage of positive cells
–
–
+
+
CD4 CD8
Percentage of positive cells
+
–
+
+
10
+
–
+
CD44 CD25
CD44 CD25
MFI = 7
MFI = 48
CD4 CD8
CD44 CD25
–
CD4 CD8
CD44 CD25
+
MFI = 29
+
CD4 CD8
–
MFI = 21
–
MFI = 16
CD44 CD25
0
10
20
30
40
50
60
0
20
40
60
80
100
Figure 2. Smo expression in adult thymocytes. (a) Flow cytometric analysis shows the highest levels of Smo receptor expression in DN (CD4–CD8–) thymocytes.
Adult thymocytes were analysed for Smo levels combined with CD4/CD8 staining and bars represent the percentage of Smo positive cells in gated subpopulations
defined by CD4/CD8 expression. Results are the mean (±SEM) of six different experiments. (b) Further analysis of isolated adult DN thymocytes according
to CD44/CD25 cell markers demonstrates the highest expression of Smo receptor in CD44+CD25+DN thymocytes decreasing in subsequent stages as DN cells
differentiate. Bars represent the percentages of positive cells (mean ± SEM), and their mean fluorescence intensities (MFI) are shown.
(DP) and CD4+CD8–/CD4–CD8+ single positive (SP) thymocyte
subpopulations (Fig. 2). The analysis of the DN thymocyte
subsets defined according to CD44 and CD25 antigen
expression shows that CD44+CD25– DN cells, including the
earliest thymic precursor cells which are not committed
to the T cell lineage(99), do not express significant levels of
Smo. In the next compartment, CD44+CD25+ pro-T cells,
Smo expression is upregulated, whereas in the subsequent
CD44–CD25+ and CD44–CD25– DN subpopulations, both
the proportions of positive cells and the levels of expression,
gradually decrease (Fig. 2). Therefore, among thymocyte
precursors, the main responders to Shh are, presumptively,
the CD25+ DN cells. This cell subset is undergoing the
rearrangement of the TCRβ chain gene that, if successful,
will allow the expression of a functional pre-TCR, which
will signal inducing both proliferation and differentiation
of T-cell precursors, as well as allelic exclusion of the TCRβ
locus(100-102).
The molecular transduction machinery of the hedgehog
cascade, including the transcriptional factors Gli1, Gli2 and
Gli3, are also expressed in the murine thymus. Interestingly,
the relative levels of gli1, gli2 and gli3 mRNA found in fetal
and adult thymus are different. Whereas in the adult thymus
gli1>gli2>gli3, inverse proportions are found in the fetal
samples, which suggests that the contribution of Hh cascade
changes during development(25).
In contrast to the murine model, all Hh proteins are
expressed in the human thymus (Sacedón et al, manuscript
in preparation). By using double immunofluorescence,
Shh expression is found to be restricted to epithelial
cells located in the subcapsullar and medullar regions,
whereas Ihh-positive and Dhh-positive epithelial cells are
scattered at random throughout the thymic cortex and
medulla (Fig. 3). The components of the Hh receptor are
expressed by both thymocytes and epithelial cells in all
thymic compartments, although Ptc is expressed by a
higher number of cells than Smo. In contrast, the expression
of Ptc2, a Ptc homologue, is restricted to epithelial cells
which appear dispersed throughout the thymic parenchyma
(Fig. 3). When the expression of the Hh receptor is analysed
in the different human thymocyte subsets, thymocyte
precursors express the highest levels of Smo, which is
downregulated in DP and SP cell subsets, as previously
shown for mice. Moreover, our results indicate a role for
Shh in increasing the survival of CD34 thymic precursors
as well as in decreasing their proliferation and differentiation
(Vicente et al, manuscript in preparation). From this
evidence a simple question is derived: Is Smo downregulation
a mere consequence of T cell differentiation or a requirement
for thymocytes to progress into T cell differentiation
program?.
The involvement of Hh in T cell differentiation was
addressed in vitro using mouse foetal thymus organ cultures
(FTOC) that were maintained for three days in the presence
of growing doses of Shh or the monoclonal antibody 5E1,
which specifically blocks Shh binding to Ptc. Such experiments
21
HEDGEHOG PROTEINS: EXPRESSION AND FUNCTION IN THE THYMUS
VOL. 22 NUM. 1/ 2003
A
B
Figure 3. Expression of Hh proteins in the human thymus. Diagram indicating
the distribution of expressed Hh proteins in the human thymus as demonstrated
by immunofluorescence. Note the restricted expression of Hh ligands (Shh,
Ihh and Dhh) and Ptc2 receptor to thymic epithelial cells while Smo and Ptc
receptors are widely distributed (Sc, subcapsule; C, cortex; M, medulla).
reveal the capacity of exogenous Shh to block thymocyte
development at the CD25+ DN stage (Fig. 4), after initiation
of TCRβ chain gene rearrangement, whereas neutralisation
of endogenous Hh leads to an earlier appearance of high
numbers of DP thymocytes (Fig. 4). These increased numbers
of DP cells are actually reflecting an acceleration of thymocyte
differentiation since there is a concomitant increase of
the more mature subpopulation of DN cells, CD44–CD25–
(Fig. 4), and because neither proliferation nor survival are
induced in the DP cell subset arising in anti-Shh treated
FTOC(25).
Although Shh neutralisation promotes differentiation
of DN to DP cells, it is unable to replace a pre-TCR signal.
This is consistent with the fact that the extra DP thymocytes
produced in the presence of the anti-Shh antibody show a
normal expression of TCRβ chain, and the inability of antiShh treatment to overcome the developmental arrest at the
CD25 + DN stage exhibited by Rag1 –/– thymocytes (25).
Nevertheless, Shh signalling abrogation does enhance the
differentiation of Rag1–/– thymocytes induced by anti-CD3
stimulation, which mimics the physiological pre-TCR
signal(25). This indicates that Shh signalling inhibition can
only promote thymocyte development after the pre-TCR
complex has instructed CD25+ DN cells to progress to DP
thymocytes.
As mentioned above, ligation of CD3, and therefore preTCR signalling, determines cell cycle progression,
differentiation from DN to DP cells and allelic exclusion of
the TCRβ locus, but also induces the downregulation of
both smo RNA and cell surface Smo expression and promotes
on developing DN thymocytes a refractory state to Hh
signalling within 2 days of receiving the pre-TCR signal(25).
22
Figure 4. Exogenously added Shh arrests thymocyte differentiation while
Shh neutralization stimulates thymocyte maturation. (A) CD4/CD8 expression
in E14 FTOC maintained in the presence either of Shh or an anti Shh
mAb. (B) Phenotype of DN (CD4–CD8–) thymocytes obtained from cultures
above described and analysed for CD44/CD25 expression.
This indicates a direct link between the pre-TCR and Hh
signalling pathways and, indirectly, points out that preTCR complex could affect the Hh signaling.
Therefore, Shh might function to maintain CD25+ DN
cells as non-proliferating cells while they are rearranging
their TCRβ chain genes, since neutralisation of Hh signalling
increases the proliferative rate of CD25+ DN cells(25). The
successful TCRβ gene rearrangement and the subsequent
expression and signalling of the pre-TCR has an immediate
consequence that is the downregulation of Smo. Consequently,
Shh can no longer signals and DN cells are released to
proliferate and differentiate to the DP cell stage. Thus,
termination of Hh signalling seems to be a pre-requisite for
differentiation of DN to DP cells(25).
The effects of Hh proteins on T cells have been recently
extended to periphery(26). CD4 T lymphocytes have been
demonstrated to produce Shh, and anti-CD3 stimulation
increases that production. Resting and stimulated CD4 T
cells express Smo and Ptc receptors as well as the transcription
factor Gli1. This expression increases after stimulation and
Hh cascade reduces apoptosis and induces cell growth after
suboptimal anti-CD3/CD28 in vitro stimulation. Neutralisation
of Shh in the cultures with high doses of 5E1 reduces cell
proliferation but not cell survival(26).
R. SACEDÓN ET AL.
INMUNOLOGÍA
Figure 5. BMP4 production in the murine thymus. Frozen sections from
mouse thymus were double stained with anti-BMP4 and anti-cytokeratin
antibodies for demonstrating the association of BMP4 production with
cytokeratin-positive epithelial cells. Positive staining is marked with short
arrows in the subcapsular (SC) epithelial cells and with long arrows in the
cortical region (C). x 300.
BMP/HH RELATIONSHIPS AND
T-CELL DIFFERENTIATION
Bi-directional influences between Bone Morphogenetic
Protein (BMP) and Hh cascades are marked during
development. BMP inhibitors, as Chordin, can sensitise
neuroepithelial cells to notochord-derived Shh, facilitating
the ability of Shh to induce the floor plate that is inhibited
by BMP family(103,104). During anlagen hair cycle epithelial
Shh acts on the follicular epithelium to promote proliferation
and down-growth of the anlagen hair follicle while BMPs
antagonise with Shh expression. Noggin secreted by dermal
papila, probably under Shh control, abrogates Shh blockade
through BMPs, that close the feedback loop inhibiting
BMPs(105). On the contrary, BMP and Ihh signalling positively
interact to co-ordinate chondrocyte proliferation and
differentiation. Ihh, produced by hiperthrophic chondrocytes,
promotes proliferation of adjacent chondrocytes and, in
addition, induces the expression of several BMP genes in
the perichondrium and in the proliferating chondrocytes.
On the other hand, BMPs activate the expression of Ihh and
negatively regulate the differentiation of terminal hyperthrophic
chondrocytes(106).
Recently, it has been proposed that Shh functions as a
regulator of pluripotent hematopoietic stem cells via
mechanisms that are dependent on downstream BMP4
signals(24). The expression of BMPs in the thymus has also
been described. BMP4 expression is found in thymic epithelial
cells located in the subcapsular and cortical areas (Fig. 5),
whereas BMP receptors are mainly expressed by thymocytes.
The specific downstream effector molecules for the BMP
signalling pathway (Smad 1, 4, 5 and 8), as well as the
extracellular inhibitors regulating BMP signalling (Noggin,
Chordin and Twisted Gastrulation) are also expressed in
the thymus(107, 108).
BMP4 inhibits thymocyte proliferation, enhances thymocyte
survival and arrests thymocyte differentiation at the
CD44+CD25– DN stage, before commitment to T cell lineage.
Conversely, neutralisation of endogenous BMP2/4 by
treatment of FTOC with the antagonist Noggin promotes
thymocyte differentiation increasing the numbers of DP
cells, although again it is unable to overcome pre-TCR
signalling, as also commented for the anti-Hh treatment(107).
Therefore, as BMP4 arrests thymocyte development at an
earlier developmental stage than Shh, it seems evident than
BMP signalling must be previous to Shh signalling in the
regulation of thymocyte development. BMP4 signalling
might be involved in the specification of T cell lineage
commitment, and the timing and speed of the differentiation
of thymocyte precursors along the T cell lineage.
However, a later influence of BMPs in thymocyte
development has been also described(108). This involves a
blockade in the progression from DN and immature single
positive (ISP) to DP stage, which is abrogated after pre-TCR
signalling that induces upregulation of Twisted gastrulation.
This protein is secreted by thymocytes and synergizes with
endogenous Chordin, which exerts similar activities as
Noggin, enhancing its capacity to block BMP actions. It is
unlikely again that Shh uses BMP4 signalling to exert its
blockade at the CD44–CD25+ cell stage, but a role of Shh in
BMP4 pathway in the thymus should be addressed.
It will be, therefore, interesting to further assess these
proposed functions of Hh and BMP4 as regulators of early
thymocyte development by analysing the relationships
between these two signalling pathways and those with other
patterning genes also functioning in the thymus.
ACKNOWLEDGMENTS
This work was supported by grants PB97-0332, PM990060, BMC2001-2025 from the Spanish Ministry of Science
and Technology and CAM08.3/0041/2000 and CAM
08.3/0018.1/2001 from the Comunidad Autónoma de Madrid.
CORRESPONDENCE TO:
Dr. Agustín G. Zapata
Departamento de Biología Celular
Facultad de Biología
Universidad Complutense
28040 Madrid, Spain.
Phone:34-91-3944979. Fax: 34-91-3944981
email: [email protected]
23
HEDGEHOG PROTEINS: EXPRESSION AND FUNCTION IN THE THYMUS
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