1 Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609,
Singapore
2 MRC Intercellular Signalling Group, Centre for Developmental Genetics, School
of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10
2TN, UK
* Author for correspondence (e-mail: s.roy{at}sheffield.ac.uk)
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Summary |
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Key words: Sonic Hedgehog, Patched, Cyclin, Proliferation, Cell cycle
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Introduction |
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Apart from its influence on the fate of cells, in many developmental
contexts, HH signalling has been associated with proliferative responses in
target cells. For example, SHH has been implicated as a crucial regulator of
growth and patterning of the cerebellum
(Dahmane and Ruiz-i-Altaba,
1999; Wallace,
1999
; Wechsler-Reya and Scott,
1999
). Here, the signal is produced and secreted by the Purkinje
neurons and appears to have a definitive mitogenic influence on the
proliferation of cerebellar granule neuron precursors (CGNPs) in the outer
granule cell layer. Similarly, during hair follicle morphogenesis in mammals,
loss of SHH, which is normally expressed at the tip of the developing
epidermal placode, dramatically reduces cell proliferation in the follicular
rudiment (Bitgood and McMahon,
1995
; Chiang et al.,
1999
; St-Jacques et al.,
1998
). Furthermore, another vertebrate Hh paralogue, Indian HH
(IHH), has a central role in cartilage formation
(St-Jacques et al., 1999
), and
recent investigations suggest that the signalling pathway is required
autonomously in the precursor chondrocytes for their proliferation
(Long et al., 2001
).
In Drosophila, as in vertebrates, HH plays an important role in
patterning the appendage primordia. During wing development, HH induces the
expression of a another morphogen, a transforming growth factor ß
(TGFß) homologue, Decapentaplegic (DPP), and is believed to regulate
pattern as well as cell proliferation largely through the activity of this
secondary signal (Burke and Basler,
1996; Martin-Castellanos and
Edgar, 2002
; Nellen et al.,
1996
). DPP is also a critical proliferative cue for cell division
in the germ line of the Drosophila ovary
(Xie and Spradling, 1998
), a
process in which the role, if any, of HH is unclear. By contrast, several
lines of evidence indicate that HH signalling is essential for the
proliferation of the ovarian somatic stem cells and, as in the case of CGNPs
and chondrocytes in vertebrates, this effect could indeed be direct
(Forbes et al., 1996
;
Zhang and Kalderon, 2001
).
Despite the prospect that, at least in certain circumstances, HH can directly trigger cell proliferation, the underlying mechanism had remained enigmatic. Recent discoveries using molecular and biochemical approaches in cultured vertebrate cells, in conjunction with genetic analysis in Drosophila, have now provided evidence that the activities of HH signalling components can indeed interface with core cell cycle regulators and modulate their expression and/or activity. We discuss new findings from disparate lines of investigation and their significance in extending our perception of the association between HH signalling and the cell cycle.
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HH signalling controls the expression cell cycle regulators |
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A hallmark of classic mitogens is that they are capable of eliciting a proliferative response even from mitotically quiescent cells that are arrested in G0 phase. Interestingly, in the above experiments, SHH was unable to make quiescent granule cell precursors re-enter the cell cycle. Thus, at least in this context, SHH activity cannot be equated entirely with that of a typical mitogen. Thus, in normal development, SHH signalling might be necessary to maintain rather than initiate CGNP proliferation in the cerebellum. It is also notable that this effect of SHH on CGNP proliferation appears to occur independently of the mitogen-activated protein (MAP) kinase activation.
Examination of the status of D-type cyclins, central regulators of G1 phase
progression, revealed that, upon incubation with SHH, the CGNPs preferentially
upregulate cyclin D1 and D2 RNA and cyclin D1 protein. The
induction of cyclin D2 protein seemed to be much more restricted in this
situation, possibly owing to some kind of post-transcriptional regulation. A
similar upregulation of cyclin D1 has also been attributed to IHH activity in
proliferating chondrocytes during cartilage development
(Long et al., 2001). Complexes
of D-type cyclins with their cognate CDKs regulate the activity of the
retinoblastoma (RB) protein, such that hyperphosphorylated RB is no longer
able to antagonise the E2F transcription factors, which results in the
expression of S-phase-promoting factors, such as cyclin E. Consistent with the
observation that SHH promotes upregulation of D-type cyclins is the
accumulation of hyperphosphorylated RB under these conditions, an effect that
can be specifically blocked by activating protein kinase A (PKA), a potent
intracellular inhibitor of HH signal transduction (see
Fig. 1). Notably, however,
primary cultures of CGNPs derived from mice lacking D1 or D2 cyclins show
wild-type proliferative responses to SHH stimulation. While this would suggest
that the D-type cyclins are functionally redundant, it is striking that
animals lacking cyclin D2 (but not cyclin D1) are characterised by reduced
numbers of granule cells in their cerebella
(Fantl et al., 1995
;
Huard et al., 1999
;
Sicinski et al., 1995
). Such a
discrepancy between the in vivo effects and the in vitro properties of mutant
cells may indicate an additional role for cyclin D2 that is distinct from its
promotion of G1 phase progression. However, the non-physiological conditions
of cell culture systems should perhaps also be taken into account. In this
context, analysis of the effects of SHH on cyclin D1 D2 double-mutant
CGNP cells, as well as exploration of the proliferative responses of
controlled SHH misexpression in the cerebella of intact cyclin D2
mutant mice, may be particularly revealing.
An independent line of evidence for the transcriptional effects of HH
signalling on D-type cyclins has come from gene expression profiling using
microarrays to probe a rat kidney cell line stably transformed with human GLI1
(Yoon et al., 2002), a
transcriptional activator of HH target genes in vertebrates
(Fig. 1). Under these
conditions, there was specific upregulation of cyclin D2 RNA, an
effect that was further confirmed through northern hybridisations. Scanning of
the genomic region upstream of the human cyclin D2 gene revealed a
consensus binding site for GLI1 within the core promoter that can be retarded
in gel shift assays in the presence of recombinant human GLI1. GLI1 is itself
a direct target of HH signalling and is induced by the activities of other GLI
proteins (see Fig. 1). This
could partly explain the requirement of protein synthesis for SHH-mediated
cyclin gene expression in cultured CGNPs (Kenny and Rowitch, 2000).
Clearly, further work will be required to provide a better understanding of
whether HH influences cyclin gene transcription only through GLI1 or
additional modes of regulation, through other GLIs or intermediate steps.
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Control of cell proliferation and cellular growth by HH in Drosophila: insights from genetic analysis |
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Duman-Scheel and colleagues showed that overexpression of the
Drosophila RB homologue in these proliferating eye cells delays their
entry into S phase and that the effect can be suppressed by loss of one copy
of the wild-type ptc gene. This link between HH signalling and a
primary cell cycle regulator is reinforced by observations that photoreceptor
precursors in the eye primordium that lack smoothened (smo)
gene function and are therefore incapable of transducing the HH signal (see
Fig. 1) fail to enter the
second mitotic wave. In addition, ectopic activation of HH signalling can
induce cells normally arrested in G1 to enter S phase precociously. These
effects of modulated HH signalling on the proliferation patterns of eye cells
are mirrored by corresponding alterations in the levels of cyclin D
and cyclin E transcripts and proteins. Thus, the control of cell
cycle in the eye by HH must be mediated in part through its regulation of
these cell cycle mediators and is consistent with previous reports describing
the ability of ectopic cyclin E to drive premature S phase entry in this
tissue (Crack et al., 2002;
Richardson et al., 1995
).
Intriguingly, Duman-Scheel et al. provide further evidence that, in this
instance, HH in fact induces cell proliferation through two independent
influences on the levels of cyclins. HH signalling not only promotes S phase
entry through the induction of cyclin D, which suppresses RB function
(and thereby activates E2F targets such as cyclin E), but also
directly stimulates transcription of cyclin E itself through Cubitus
interruptus (CI), the GLI family protein that activates HH target genes in
flies.
Cell proliferation and cell growth are important determinants of the size
and shape of developing embryos, organs and tissues. The extent to which these
two processes are connected and coordinated is one of the central focuses in
developmental biology (for a review, see
Tapon et al., 2001). Evidence
for such a link comes from the finding that cyclin D has the capacity to drive
both cell division and cellular growth
(Datar et al., 2000
;
Meyer et al., 2000
). Likewise,
HH signalling is known to shape growth and pattern of a variety of tissues
during development. Consistent with this scenario is the fact that clones of
cells in the proliferating wing primordium exhibit enhanced growth in the
presence of unabated HH signalling, as opposed to decreased growth when the
pathway is constitutively repressed
(Duman-Scheel et al., 2002
).
Furthermore, in line with the ability of HH signalling to induce cyclin
D expression, these effects of HH on the growth of developing wing cells
are critically dependent on the activity of this cyclin and its associated
kinase, CDK4.
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PTC1 binds the M-phase promoting factor (MPF) and regulates its activity |
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Taken together, this study suggests that apart from regulating the
expression of cyclins, HH may have a more immediate influence on the cell
cycle through regulation of the PTC-mediated subcellular localisation of the
MPF. Although the data supporting this idea are biochemically robust, they are
based to a large extent on overexpression studies in cell culture and not only
beg corroboration in an in vivo developmental context but also provoke further
challenging questions about the cellular basis of this interaction. Perhaps
the most perplexing of them involves the subcellular compartment in which the
interaction is likely to occur, especially given the mixed opinion in the
literature about the distribution of PTC within the cell. Studies of
Drosophila PTC in vivo, as well as in cultured cells that express the
endogenous protein, have revealed that it has a largely intracellular
localisation in multivesicular bodies
(Capdevila et al., 1994;
Denef et al., 2000
;
Strutt et al., 2001
). On the
other hand, overexpression of mammalian PTC1 in cell culture, including the
data presented by Barnes and colleagues, have shown that PTC1 `atypically'
decorates the plasma membrane (Carpenter et
al., 1998
; Stone et al.,
1996
) or, in common with Drosophila PTC, is present in
cytoplasmic vesicles (Incardona et al.,
2002
). Furthermore, given the caveat that even conserved regions
of the PTC molecule can have distinct behaviours in different species
(Johnson et al., 2002
), it
will be important to determine the integrity of this interaction in other
systems.
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Conclusion: HH, cell cycle and cancer |
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Clearly, these are the beginnings of our appreciation of how HH influences the cell cycle and cell growth, and the studies discussed here should serve to reinforce how little we know about this important aspect of HH function. Nevertheless, taking into consideration the morbid effects of unrestrained HH signalling, these new data are particularly significant. We have to wait with eager anticipation for the emergence of a more lucid picture that integrates all these recent findings and for the identification of other ways by which HH can control such processes and contribute to carcinogenesis in situations of inappropriate signalling activity.
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Acknowledgments |
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