Arginine refolds, stabilizes, and restores function of mutant pVHL proteins in animal model of the VHL cancer syndrome
The von Hippel-Lindau (VHL) syndrome is a rare inherited cancer, caused by mutations in the VHL gene, many of which render the VHL protein (pVHL) unstable. pVHL is a tumor-suppressor protein implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase complex which targets the hypoxia-inducible factor (HIF) for degradation. Previous analyses have shown that common oncogenic VHL mutations render pVHL less stable than the wild-type protein, distort its core domain and as a result reduce the ability of the protein to bind its target HIF-1alpha. Among various chemical chaperones tested, arginine was the most effective in refolding mutant of pVHL. This study examined the consequences of administering L- or D-arginine to a Drosophila VHL model and to human renal carcinoma cells, both expressing misfolded versions of human pVHL. Arginine treatment increased pVHL solubility in both models and increased the half-life of the mutant pVHL proteins in the cell culture. In both models, L- as well as D-arginine enhanced the ability of wild-type pVHL and certain misfolded mutant versions of pVHL to bind ODD, the HIF-derived target peptide, reflecting restoration of pVHL function. Moreover, continuous feeding of Drosophila expressing misfolded versions of pVHL either L- or D-arginine rich diet rescued their lethal phenotype. Collectively, these in vivo results suggest that arginine supplementation should be examined as a potential novel treatment for VHL cancer syndrome (Shmueli, 2018).
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Delgehyr, N., Wieland, U., Rangone, H.,
Pinson, X., Mao, G., Dzhindzhev, N.S., McLean, D., Riparbelli,
M.G., Llamazares, S., Callaini, G., Gonzalez, C. and Glover,
D.M. (2012). Drosophila Mgr, a Prefoldin
subunit cooperating with von Hippel Lindau to regulate tubulin
stability. Proc Natl Acad Sci U S A 109: 5729-5734. PubMed ID: 22451918
Abstract
Mutations in Drosophila merry-go-round (mgr)
have been known for over two decades to lead to circular mitotic
figures and loss of meiotic spindle integrity. However, the
identity of its gene product has remained undiscovered. This study
shows that mgr encodes the Prefoldin subunit counterpart
of human von Hippel Lindau binding-protein 1. Depletion of Mgr
from cultured cells also leads to formation of monopolar and
abnormal spindles and centrosome loss. These phenotypes are
associated with reductions of tubulin levels in both mgr
flies and mgr RNAi-treated cultured cells. Moreover, mgr
spindle defects can be phenocopied by depleting β-tubulin,
suggesting Mgr function is required for tubulin stability.
Instability of β-tubulin in the mgr larval brain is
less pronounced than in either mgr testes or in cultured
cells. However, expression of transgenic β-tubulin in the
larval brain leads to increased tubulin instability, indicating
that Prefoldin might only be required when tubulins are
synthesized at high levels. Mgr interacts with Drosophila
von Hippel Lindau protein (Vhl). Both proteins interact with
unpolymerized tubulins, suggesting they cooperate in regulating
tubulin functions. Accordingly, codepletion of Vhl with Mgr gives
partial rescue of tubulin instability, monopolar spindle
formation, and loss of centrosomes, leading to a proposed
requirement for Vhl to promote degradation of incorrectly folded
tubulin in the absence of functional Prefoldin. Thus, Vhl may play
a pivotal role: promoting microtubule stabilization when tubulins
are correctly folded by Prefoldin and tubulin destruction when
they are not (Delgehyr, 2012).
Highlights
- Mgr is a subunit of the highly conserved Gim
complex/Prefoldin.
- Spindle abnormalities result from tubulin destabilization
following Mgr depletion.
- Levels of free αβ-tubulin sensitize Mgr activity.
- Mgr and Vhl cooperate to control the degradation of
αβ-tubulins.
Discussion
The finding that the Drosophila merry-go-round
gene encodes a subunit of the Prefoldin complex accounts for
aberrant structure and function of spindles and centrosomes in
cells depleted of its gene product. The inability to correctly
fold tubulins in Prefoldin-deficient cells leads to tubulin
instability and, hence, defects that can be phenocopied by
depleting β- or γ-tubulin. However, whereas
β-tubulin depletion phenocopied all of the defects observed,
γ-tubulin depletion only recapitulates some of them. The
more dramatic phenotypes seen in Mgr-deficient cells expressing
high levels of tubulin (primary spermatocytes and neuroblasts
expressing a β-tubulin transgene) suggest that the Prefoldin
complex is critical to maintain tubulin levels above a certain
threshold of tubulin expression. This finding could be a
consequence of the impact of an excess of tubulin upon its complex
folding pathway. Interestingly, in mammalian cells, increased
soluble tubulin, in response to a MT-destabilizing agent, leads to
the rapid degradation of tubulin. In Drosophila,
tubulins in the testes are the most affected by the absence of Mgr
compared with other tissues. Indeed, it may be of particular
importance to regulate tubulin levels at the late stages of
spermatogenesis, where the very large meiotic cells are provided
with proportionally large amounts of tubulin that are used in the
meiotic spindle but have a major additional purpose: the building
of the sperm tail. Similarly, in the mouse, the effects of
depletion or mutation of prefoldin subunits are largely restricted
to the brain, where tubulin levels are also very high. Whether
this tissue specificity is a consequence of tubulin levels will be
an interesting question to address. Finally, the demonstration
that Vhl is required for tubulin destruction in the absence of Mgr
and the ability of Vhl to interact with tubulin monomers and
dimers raises the possibility that its role as an E3
ubiquitin-protein ligase could come to play in regulating tubulin
levels (Delgehyr, 2012).
The idea that Vhl and Prefoldin can cooperate in regulating
protein stability has been raised earlier by another study that
identified the prefoldin subunit VBP1 as a binding partner of the
HIV-1 viral integrase and suggested it to mediate the interaction
of the integrase with the Cul2-Vhl E3-Ubiquitin ligase. This
finding led to the suggestion for a role for prefoldin at a
pivotal part of the pathway that would determine whether a protein
was passed on to the CCT chaperonin for folding or to the
proteasome for degradation. Similarly, this study speculates that
prefoldin as a partner of Vhl may well serve a key role in
regulating the equilibrium between tubulin targeted for
destruction or for folding and incorporation into MTs. The
concentration of assembly-competent tubulin must be tightly
controlled because it affects cytoskeletal dynamics. Vhl might
contribute to this influence by an effect on MT dynamics through
interaction with MAPs on the MT lattice and by intervening in the
regulation of tubulin folding. There is growing evidence for a
critical function of Vhl in stabilizing cytoplasmic MTs and
axonemal MTs in response to levels of soluble tubulin.
Reciprocally, MT stability can contribute to regulating levels of
proteins that are targets of the Cul2-Vhl E3-Ubiquitin ligase,
such as the HIF proteins, the levels of which fall when their
mRNAs accumulate in cytoplasmic P-bodies for translational
repression following MT disruption. It will be important in future
to consider the roles played by the Prefoldin complex and Vhl to
understand the interrelationships between the machinery regulating
tubulin levels in relation to MT stability, both in normal and
tumor cells (Delgehyr, 2012).
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Hsouna, A., Nallamothu, G., Kose, N.,
Guinea, M., Dammai, V. and Hsu, T. (2010). Drosophila
von Hippel-Lindau tumor suppressor gene function in epithelial
tubule morphogenesis. Mol Cell Biol 30: 3779-3794. PubMed ID: 20516215
Abstract
Mutations in the human von Hippel-Lindau (VHL)
gene are the cause of VHL disease that displays multiple benign
and malignant tumors. The VHL gene has been shown to
regulate angiogenic potential and glycolic metabolism via its E3
ubiquitin ligase function against the alpha subunit of
hypoxia-inducible factor (HIF-α). However, many
HIF-independent functions of VHL have been identified. Earlier
studies also indicate that the canonical function cannot fully
explain the VHL mutant cell phenotypes, although it is still
unclear how many of these noncanonical functions relate to the
pathophysiological processes because of a lack of tractable
genetic systems. This study reports the first genomic mutant
phenotype of Drosophila melanogaster VHL (dVHL)
in the epithelial tubule network, the trachea, and shows that dVHL
regulates branch migration and lumen formation via its endocytic
function. The endocytic function regulates the surface level of
the chemotactic signaling receptor Breathless (Btl) and promotes
clearing of the lumen matrix during maturation of the tracheal
tubes. Importantly, the regulatory function in tubular
morphogenesis is conserved in the mammalian system, as conditional
knockout of Vhl in mouse kidney also results in similar cell
motility and lumen phenotypes (Hsouna, 2010).
Highlights
- Characterization of the dVHL1 allele.
- The dVHL loss-of-function phenotype results in
abnormal tracheal structure during embryonic development.
- dVHL1 germ line mutants show complete failure in
forming trachea.
- dVHL mutant tracheal cells exhibit ectopic migration
phenotypes.
- dVHL mutants display overactive FGFR/Btl signaling.
- dVHL protein interacts with abnormal
wing discs (awd).
- Vhl knockout in mouse kidney tubules generates lumen
and branching defects.
Discussion
This study shows that dVHL regulates tracheal tubule
development in two aspects—branch migration and lumen
formation. These two aspects of tracheal morphogenesis are
regulated by the same endocytic function; one involves
internalization of the Btl signaling receptor, and the other
involves resorption of lumen materials (Hsouna, 2010).
The study also shows that dVHL genetically interacts
with endocytic pathway components such as awd, shibire
(shi) and Rab5
in both branch migration and lumen formation phenotypes but
interacts with btl signaling pathway components only in
branch migration phenotypes. The lumen defects in the dVHL
mutant are similar to those found in the wurst
mutant. wurst encodes a transmembrane protein that
promotes clathrin-mediated endocytosis of lumen material. wurst
function is specific for lumen maturation and, unlike dVHL,
has little effect on branch migration. This indicates that wurst
may be specific for pinocytosis (uptake of extracellular
materials) and not involved in internalization of surface
proteins, while the dVHL- and awd-regulated
dynamin-Rab5 pathway is necessary for both (Hsouna, 2010).
It was earlier reported that some of the tracheal tubule
migration defects generated by dVHL RNA interference could be
attributed to upregulation of Sima, the Drosophila
homolog of HIF-α, which in turn upregulates btl
transcription. Hypoxia-induced, Sima-dependent btl
transcription has also been demonstrated in terminal branching in
the larval trachea. It is possible that the endocytic and the
HIF-dependent functions of VHL are not mutually exclusive. On the
other hand, it has been shown that the stereotypic tracheal branch
migration pattern in the embryo (as opposed to the terminal
branching in larvae) is normally not dependent on sima
function. Also, overexpression of the wild-type btl
transgene in the embryonic trachea using the cognate btl
promoter, although exhibiting ectopic elevation of the btl
transcript level, could not lead to embryonic tracheal defects.
This indicates that posttranscriptional regulation is the major
mechanism for modulating active Btl level and thus Btl signaling.
Indeed, it was shown in this study that the exogenously expressed
Btl::GFP protein is under stringent control at the protein level.
Also, sima can only modestly suppress the dVHL1
branch migration phenotype. It is interesting to point out that a
robust rescue of phenotypes in the btl promoter-driven dVHL
RNA duplex by sima heterozygotes has been shown earlier.
This may be because the exogenous btl promoter,
presumably positively regulated by sima, is itself
downregulated in the sima mutant, thereby diminishing
the dVHL duplex expression, resulting in apparent rescue
(Hsouna, 2010).
It should be noted that primary and secondary tracheal tubule
development during embryogenesis is a stereotypic, genetically
programmed process. Tissue microenvironmental factors such as
hypoxia do not play a significant role in this process. On the
other hand, in late embryonic and larval stages, when the trachea
system extends to connect with internal organs, localized hypoxia
in the target tissue is critical in inducing FGF/bnl
expression and thus promoting terminal branching. HIF-α/Sima
has been shown to also play an important role in the terminal
tracheal tubule cells in sensing hypoxia and inducing FGFR/btl
expression. This mechanism, in the absence of dVHL
mutation, can contribute to tracheal branching probably because it
is sensitized by the overproduced Bnl ligand in hypoxic
conditions. It is conceivable that dVHL loss of function
during embryogenesis can result in similar upregulation of the btl
gene transcription. However, results in this study indicate that
in nonhypoxic development of trachea in the embryonic stage, the
additional function of dVHL in regulated protein internalization
plays the major role in tracheal morphogenesis (Hsouna, 2010).
Most interestingly, the study demonstrates that the ectopic
branching of the tubule epithelial cells and the malformation of
lumen phenotypes are reproducible in the kidney tubules of mice
with conditional Vhl knockout and in organoid culture
using primary tubule cells. This is highly significant, since
dilated tubules (minicysts) have been documented as preceding
renal cell carcinoma. Thus, these findings provide a plausible
mechanistic explanation, involving increased cell motility and
disruption of tubule epithelium, for the etiology of VHL mutant
kidney cancer. In addition, another study implicated dVHL
in the morphogenesis of organ-associated epithelium, the follicle
cells in the ovary. This function is mediated by another
HIF-independent activity of dVHL that stabilizes
microtubule bundles. Future studies should exploit further the Drosophila
genetic system for elucidating how various VHL functions and a
myriad of disease-related VHL mutations may differentially affect
the pathophysiological roles of this interesting tumor suppressor
gene (Hsouna, 2010).
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Duchi, S., Fagnocchi, L., Cavaliere,
V., Hsouna, A., Gargiulo, G. and Hsu, T. (2010).
Drosophila VHL tumor-suppressor gene regulates
epithelial morphogenesis by promoting microtubule and aPKC
stability. Development 137: 1493-1503. PubMed ID: 20388653
Abstract
Mutations in the human von Hippel-Lindau (VHL) genes are
the cause of VHL disease, which displays multiple benign and
malignant tumors. The VHL gene has been shown to
regulate angiogenic potential and glycolic metabolism via its E3
ubiquitin ligase function against the alpha subunit of
hypoxia-inducible factor (HIF). However, many other
HIF-independent functions of VHL have been identified and there is
evidence that the canonical function cannot fully explain the VHL
mutant cell phenotypes. Many of these functions have not been
verified in genetically tractable systems. Using an established
follicular epithelial model in Drosophila, this study
shows that the Drosophila VHL gene is involved
in epithelial morphogenesis via stabilizing microtubule bundles
and aPKC. Microtubule defects in VHL mutants lead to
mislocalization of aPKC
and subsequent loss of epithelial integrity. Destabilizing
microtubules in ex vivo culture of wild-type egg chambers can also
result in aPKC mislocalization and epithelial defects.
Importantly, paclitaxel-induced stabilization of microtubules can
rescue the aPKC localization phenotype in Drosophila VHL
mutant follicle cells. These results establish a developmental
function of the VHL gene that is relevant to its
tumor-suppressor activity (Duchi, 2010).
Highlights
- Drosophila VHL mutant follicle cells
exhibit epithelial defects.
- Altered epithelial marker expression in VHL mutant
cells.
- Homozygous VHL1 egg chambers show severe epithelial
defects.
- VHL functions via stabilizing microtubules (MTs).
- VHL regulates MTs and aPKC stability.
- A disease-related VHL mutant defective in MT stabilization
cannot rescue the aPKC localization phenotype.
Discussion
This study shows that Drosophila VHL is
important for establishing and maintaining epithelial integrity
via its regulation of microtubule (MT) and aPKC stability. MT
disruption and epithelial phenotypes early in oogenesis were
observed. This indicates that MT bundles in developing epithelial
cells are crucial for epithelial development and are under
pressure from dynamic instability. Without stabilizing activity
provided by VHL, MTs are disorganized and ultimately disintegrate,
resulting in loss of epithelial integrity. It was shown that
disrupted MTs interfere with proper localization of aPKC, which in
turn leads to mislocalization of downstream epithelial markers and
epithelial defects. Ex vivo experiment also demonstrates that
epithelial defects can occur within a short time (relative to the
entire oogenesis time frame) after destabilizing MTs in
non-proliferating epithelial cells. This indicates that the
maintenance of epithelial integrity is a dynamic and continuous
process even in a stable epithelium, for which MTs are crucially
important. Earler studies using RNA interference-mediated
knockdown demonstrate a morphogenic role of VHL in
trachea development. The tracheal phenotypes appear to be the
result of elevated cell motility and ectopic chemotactic
signaling. Therefore, the tracheal function of VHL might
be mediated via different VHL targets in a tissue-specific
context. Alternatively, regulation of MT stabilization might also
be the underlying mechanism. This study favors a separate,
tissue-specific function for VHL as the tracheal defects
in VHL knockdown can be relieved by decreased expression
of breathless, which encodes the chemotactic signaling
receptor in the trachea. The two VHL functions, however, are not
necessarily mutually exclusive. These different organ systems
might in the future serve as a model for testing whether the
various functions assigned to VHL are tissue-specific and
context-dependent (Duchi, 2010).
Human VHL has been shown to translocate aPKC to MTs, thereby
influencing MT reorganization. This study shows that the aPKC
mutant can affect MT organization but not stability, whereas VHL
can influence both. Conversely, disruption of MTs alone can result
in aPKC mislocalization resembling that observed in VHL
mutant cells. Importantly, paclitaxel-induced MT stabilization can
rescue aPKC localization in VHL mutant follicle cells.
The study therefore concludes that a major function of VHL
in the follicular epithelium is regulation of MT stability. Loss
of MTs leads to aPKC mislocalization and degradation. Conversely,
part of the VHL epithelial functions might be mediated
by its direct effect on aPKC stability, as exogenously expressed
aPKC-GFP fusion protein can partially rescue (not statistically
significant) the VHL mutant phenotype. Indeed, VHL can
co-immunoprecipitate with tubulin or aPKC, and that, at least in
S2 cells, aPKC levels can be affected by VHL levels without
affecting tubulin. Taken together, it appears that the epithelial
function of VHL is mediated through stabilization of MT,
with an auxiliary role in directly stabilizing aPKC (Duchi, 2010).
It has been suggested that VHL interacts with MTs via the kinesin
2 family of motors. Future studies using the epithelial system
should also address this issue in vivo. Also interestingly, it was
shown that the VHLYH mutant (single amino acid
substitution mutation of tyrosine to histidine at position 51,
equivalent to human Y98) can associate with MT but has little
MT-stabilizing activity. This suggests that the VHLYH
mutant might be defective in recruiting other proteins, possibly
including aPKC, that are important for regulating MT functions. In
light of the role of Drosophila VHL in
regulating MT stability, a function presumably important for all
cells, it is curious that the tissue-specific btl-driven
VHL expression can rescue the homozygous lethality of VHL1
(generated by replacing the wild-type copy, via homologous
recombination, with a deletion that removes 81 codons encompassing
the first two in-frame AUGs). It has been shown that tracheal
defects are the major embryonic phenotype observed in VHL
mutant. In the course of attempting to rescue the tracheal
phenotype with btl-driven VHL, rescued
homozygous adults are present. This indicates that the MT
stabilizing function of VHL is not required in all
tissues. It is possible that although VHL can enhance MT
stability, by itself it is not an essential factor for MT
polymerization. As such, some tissues might be less dependent on
VHL levels. In the follicular environment, MT rearrangement,
including depolymerization and repolymerization, is crucial when
the entire epithelial sheet moves over the germ cell complex while
the cells grow increasingly columnar. MT stabilization facilitated
by VHL might be of particular importance during this process
(Duchi, 2010).
The best-documented function of VHL is its E3 ubiquitin ligase
activity that targets the alpha subunit of the HIF transcription
factor. This activity provides an elegant mechanistic explanation
for the hypervascularity of many of the VHL tumors and for a
potential contributor to the metabolic switch to glycolysis, as
HIF can upregulate pro-angiogenic factors such as
vascular-endothelial growth factor and components in the glucose
metabolic pathway. However, there is also evidence that VHL is a
multifunctional protein. It can function as a regulator of matrix
deposition, integrin assembly, endocytosis, kinase activity,
senescence, protein stabilities and tight junction formation,
among many others. Whether tight junction disassembly in VHL
mutant cells is HIF-dependent is still unresolved; however, other
– HIF-independent – functions appear to facilitate
protein stability or activity instead of destabilizing them as a
ubiquitin ligase. Such chaperon/adaptor function has also been
implicated in promoting stability of MTs. The MT-stabilizing
function, although potentially highly significant, has so far only
been linked to cilium biogenesis and mitotic spindle orientation
in cultured RCC and renal tubule cells. The physiological and
developmental significance of this function has not been
elucidated in vivo. Indeed, it is unclear how loss of many of
these HIF-independent functions contributes to VHL tumor formation
because of a lack of tractable genetic models (Duchi, 2010).
One crucial element in tumorigenesis is the breakdown of
epithelial integrity that ultimately leads to
epithelial-to-mesenchymal transition. This report provides the
first demonstration of a potential tumor-suppressor function for VHL
in regulating epithelial morphogenesis via its role in promoting
MT stability. Future studies should exploit further this genetic
system for elucidating how a myriad of disease-related VHL
point mutations might differentially influence such function
(Duchi, 2010).
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Doronkin, S., Djagaeva, I., Nagle,
M.E., Reiter, L.T. and Seagroves, T.N. (2010).
Dose-dependent modulation of HIF-1alpha/sima controls the
rate of cell migration and invasion in Drosophila ovary
border cells. Oncogene 29: 1123-1134. PubMed ID: 19966858
Abstract
This study analyses the role of the hypoxic response during
metastasis in migrating border cells of the Drosophila
ovary. Acute exposure to 1% O2 delays or blocks border cell
migration (BCM), whereas prolonged exposure results in the first
documented accelerated BCM phenotype. Similarly, manipulating the
expression levels of sima, the Drosophila
hypoxia-inducible factor (HIF)-1α ortholog, reveals that
Sima can either block or restore BCM in a dose-dependent manner.
In contrast, over-expression of Vhl (Drosophila
von Hippel–Lindau) generats a range of phenotypes, including
blocked, delayed and accelerated BCM, whereas over-expression of hph
(Drosophila HIF prolyl hydroxylase) only accelerates BCM.
Mosaic clone analysis of sima or tango
(HIF-1β ortholog) mutants reveals that cells lacking Hif-1
transcriptional activity are preferentially detected in the
leading cell position of the cluster, resulting in either a delay
or acceleration of BCM. Moreover, in sima mutant cell
clones, there is reduced expression of nuclear slow
border cells (Slbo) and basolateral DE-cadherin,
proteins essential for proper BCM. These results show that Sima
levels define the rate of BCM in part through regulation of Slbo
and DE-cadherin, and suggest that dynamic regulation of Hif-1
activity is necessary to maintain invasive potential of migrating
epithelial cells (Doronkin, 2010).
Highlights
- Vhl/VHL over-expression causes pleiotropic
defects.
- DE-cadherin levels are modulated by Vhl and sima.
Discussion
In addition to other investigations, this study also investigated
whether increasing Vhl expression, which would also be expected to
decrease accumulation of Sima, would affect BCM. Stocks were
created in which expression of full-length, FLAG epitope-tagged Vhl
or human VHL was driven in border cells. A broad
spectrum of phenotypes was observed ranging from normal to
accelerated to blocked migration using either driver. Compared
with wild-type chambers dissected at stage 10, in which migration
was complete, up to 40% of egg chambers show a delay in migration.
However, up to 10% of chambers exhibit accelerated migration,
moving beyond the border between the nurse cells and the oocyte.
Although 50–60% of UAS–Vhl egg chambers
display normal BCM, a small fraction (7%) show complete migration
failure. Similar results were obtained for the c522–GAL4
driver at stages 9 and 10 (Doronkin, 2010).
It is noteworthy that ectopic expression of human VHL
in the Drosophila ovary using either the slbo-GAL4 or c522-GAL4
driver also produces a similar range of phenotypes. VHL
over-expression causes delays in migration of 39/29% of chambers,
blocks migration in 7/6% of chambers, and accelerates migration in
5/8% of egg chambers, respectively. Together, these data
demonstrate that BCM is more sensitive to ectopic expression of
Sima than Vhl and provide additional evidence that the VHL pathway
is highly conserved in Drosophila. Finally, expression
of either Vhl or Hph increases BCM without damaging the cortical
cytoskeleton. In a slbo–GAL4>UAS–Vhl
egg chamber exhibiting the accelerated phenotype, the abnormal,
posterior location of accelerated clusters is consistently
accompanied by a stretching, but not complete disruption, of the
oocyte/nurse cell actin network (Doronkin, 2010).
One of the key downstream effector molecules in collective BCM is
Drosophila E-cadherin (DE-cadherin; encoded by shotgun).
The VHL/HIF axis has been shown to regulate E-cadherin levels in
human renal cancer cells. This study compared DE-cadherin
expression levels by immunostaining fixed egg chambers dissected
from UAS–sima, UAS–Vhl or sima
mosaic clone border cell clusters. In wild-type clusters,
DE-cadherin accumulates to the highest levels at the interface
between the border cells and the polar cells, located at the
center of the cluster. Accumulation is less pronounced between the
cluster and the nurse cells. In UAS–sima clusters,
expression of DE-cadherin is enhanced compared with control
clusters at the boundaries between individual border cells.
Similarly, in delayed UAS–Vhl clusters,
DE-cadherin appears to more strongly accumulate at the interface
of the cluster and the surrounding nurse cells rather than between
the border cells and polar cells. Finally, in all accelerated UAS–Vhl
clusters, DE-cadherin expression is potently reduced. It is
possible that increased accumulation of DE-cadherin in the border
cell clusters in response to Sima over-accumulation may have
strengthened cell–cell adhesions leading to stalled
migration. Similarly, reduction of the DE-cadherin levels in sima-mutant
cells could have produced weaker contacts between the neighboring
nurse cells and the migrating border cells, which would explain
the accelerated cell migration observed in UAS–Vhl
expressing cells (Doronkin, 2010).
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Mortimer, N.T. and Moberg, K.H.
(2009). Regulation of Drosophila embryonic
tracheogenesis by dVHL and hypoxia. Dev Biol 329:
294-305. PubMed ID: 19285057
Abstract
The tracheal system of Drosophila melanogaster is an
interconnected network of gas-filled epithelial tubes that
develops during embryogenesis and functions as the main
gas-exchange organ in the larva. Larval tracheal cells respond to
hypoxia by activating a program of branching and growth driven by
HIF-1α/sima-dependent expression of the breathless
(btl) FGF receptor. By contrast, the ability of the
developing embryonic tracheal system to respond to hypoxia and
integrate hard-wired branching programs with sima-driven
tracheal remodeling is not well understood. This study shows that
embryonic tracheal cells utilize the conserved ubiquitin ligase dVHL
to control the HIF-1 α/sima hypoxia response
pathway, and identifies two distinct phases of tracheal
development with differing hypoxia sensitivities and outcomes: a
relatively hypoxia-resistant ‘early’ phase during
which Sima activity conflicts with normal branching and stunts
migration, and a relatively hypoxia-sensitive ‘late’
phase during which the tracheal system uses the dVHL/sima/btl
pathway to drive increased branching and growth. Mutations in the
archipelago
(ago) gene, which antagonizes btl
transcription, re-sensitize early embryos to hypoxia, indicating
that their relative resistance can be reversed by elevating
activity of the btl promoter. These findings reveal a second type
of tracheal hypoxic response in which Sima activation conflicts
with developmental tracheogenesis, and identify the dVHL
and ago ubiquitin ligases as key determinants of hypoxia
sensitivity in tracheal cells. The identification of an early
stage of tracheal development that is vulnerable to hypoxia is an
important addition to models of the invertebrate hypoxic response
(Mortimer, 2009).
Highlights
- Stage-specific effects of hypoxia on embryonic tracheogenesis.
- dVHL is required to suppress the tracheal hypoxic response.
- dVHL genetically antagonizes sima in the
embryonic trachea.
- dVHL suppresses btl expression in the
embryo.
- dVHL and ago synergize to control embryonic
tracheogenesis
Discussion
Hypoxia-induced remodeling of tracheal terminal cells represents
the response of a developed larval tracheal system to reduced
levels of O2 in the environment. By contrast, the response of the
developing embryonic tracheal system to systemic hypoxia has not
been as well characterized. In light of the observation that
embryonic tracheal cells display hypoxia-induced activation of a
Sima-reporter and that sima promotes btl
expression in larval tracheal cells, embryonic exposure to hypoxia
may thus produce a situation in which hard-wired btl/bnl
patterning signals in the embryo come into conflict with the type
of sima/btl-driven plasticity of tracheal cell branching
seen in the larva. This study examines the effect of hypoxia on
embryonic tracheal branching and migration. It was found that
hypoxia has dramatic effects on the patterns of morphogenesis of
the primary and secondary tracheal branches. Surprisingly, varying
the timing and severity of hypoxic challenge is able to shift the
outcome from severely stunted tracheal branching to excess branch
number and enhanced branch growth. Genetic and molecular data
indicate that both classes of phenotypes, stunting and overgrowth,
involve regulation of sima activity and btl
transcription by dVHL, and that the effects of hypoxia
on tracheal development can be mimicked in normoxia by
tracheal-specific knockdown of dVHL. This observation
confirms a central role for dVHL in restricting the
hypoxic response in vivo, and identifies a role for dVHL
as a required inhibitor of sima and btl during
normal tracheogenesis (Mortimer, 2009).
Since Trh and Sima/HIF-1α share a similar consensus DNA
binding site, it is likely that the tracheal phenotypes elicited
by either hypoxia or dVHL knockdown are to some degree
the product of a combined ‘Trh/Sima-like’
transcriptional activity in tracheal cells. This conclusion is
supported both by the general phenotypic similarity (i.e.
migration and overgrowth defects) between hypoxia/dVHL
knockdown and trh
overexpression, by the modest ability of trh alleles to
suppress dVHLi phenotypes, and by the overlap of
transcriptional activity between Trh and human HIF-1α.
Indeed, Trh is well-established as a required activator of
developmental btl expression. However, because the
excess Btl activity that occurs in hypoxia or in the absence of dVHL
occurs independently of a change in Trh expression, it thus
appears to be mediated largely by increased sima
activity (Mortimer, 2009).
This study suggests that there are two distinct developmental
‘windows’ of embryogenesis during which hypoxia has
opposite effects on tracheal branching. The first corresponds to a
period immediately before and during primary branch migration that
is relatively insensitive to hypoxia. Embryos in this stage show a
minimal response to 1% O2, but show a nearly complete arrest of
migration in 0.5% O2. Interestingly, a prior study found that
similarly staged embryos (stage 11) respond to complete anoxia by
prolonged developmental arrest, from which they can emerge and
resume normal development. These somewhat paradoxical results
— that acute hypoxia is more detrimental to development than
chronic anoxia — might be explained by the observation that
chronic exposure to low O2 induces Sima activity throughout the
embryo while acute exposure activates Sima only in tracheal cells.
The former scenario may result in coordinated developmental and
metabolic arrest throughout the organism, while in the latter
scenario developmental patterns of gene expression in non-tracheal
cells may proceed such that tracheal cells emerging from an
‘early’ hypoxic response find an embryonic environment
in which developmentally hard-wired migratory signals emanating
from non-tracheal cells have ceased (Mortimer, 2009).
The second type of tracheal response occurs during a later
‘window’ of embryogenesis after btl/bnl-driven
primary and secondary branch migration and fusion are largely
complete. It involves sinuous overgrowth of the primary and
secondary branches, and duplication of secondary branches. As in
the ‘early’ response, ‘late’ hypoxic
phenotypes are controlled by the dVHL/sima pathway, yet
unlike the ‘early’ response, these phenotypes occur at
high penetrance even at 1% O2. Thus the ‘late’
embryonic tracheal system is relatively sensitized to hypoxia and
responds with increased branching in a manner similar to larval
terminal cells. Indeed, much as larval branching increases with
decreasing O2 levels, it was observed that dorsal trunk growth in
the late embryo is graded to the degree of hypoxia. The mechanism
underlying the differential sensitivity of the ‘early’
and ‘late’ tracheal system may be quite complex.
However, it was found that tracheogenesis can be sensitized to
hypoxia by reducing activity of ago, a ubiquitin ligase
component that restricts btl transcription in tracheal
cells via its role in degrading the Trh transcription factor.
Increasing transcriptional input on the btl promoter
thus appears to sensitize ‘early’ tracheal cells to
hypoxia. As Sima also controls btl transcription, one
explanation of the difference in sensitivity between different
embryonic stages may thus lie in differences in the activation
state of the btl promoter. If so then the activity of
the endogenous btl regulatory network may be an
important determinant of the threshold of hypoxia required to
elicit changes in tracheal architecture (Mortimer, 2009).
An organism can have its hypoxic response triggered in two ways,
either by systemic exposure of the whole organism to a reduced O2
environment or by localized hypoxia produced by increased O2
consumption in metabolically active tissues. Data from this study
and others suggests there may be distinctions between these two
triggers. Exposing larvae or embryos to a systemic pulse of
hypoxia results in a ‘btl-centric’ response
specifically in tracheal cells. Outside of an ‘early’
vulnerable period which corresponds to embryonic branch migration
and fusion, elevated Btl activity in embryonic tracheal cells
promotes branch duplications and overgrowth similar to that seen
in larvae. By contrast, tracheal growth induced by localized
hypoxia in the larva has been suggested to involve a ‘bnl-centric’
model in which the hypoxic tissue secretes Bnl and recruits new
tracheal branching. Whether this type of mechanism operates in
embryos, or whether embryos ever experience localized hypoxia in
non-tracheal cells, has not been established (Mortimer, 2009).
Data from this study also indicate that dVHL is a
central player in the hypoxic response pathway in embryonic and
larval tracheal cells. A prior study found that injection of dVHL
dsRNA into syncytial embryos disrupts normal tracheogenesis, but
was technically limited in its ability to conduct a detailed
analysis of dVHL function in development and
homeostasis. This study shows that dVHL knockdown
specifically in tracheal cells mimics the effect of systemic
hypoxia on embryonic tracheal architecture and larval terminal
cell branching. dVHL knockdown thus phenocopies loss of
the HPH gene fga, which normally functions to target
Sima to the dVHL ubiquitin ligase in normoxia. Moreover,
all phenotypes that result from reduced dVHL expression
can be rescued by reducing sima activity, suggesting
that Sima is the major target of dVHL in the tracheal
system. These data support a model in which dVHL, fga,
and sima function as part of a conserved
VHL/HPH/HIF-1α pathway to control tracheal morphogenesis in
embryos and larvae. The btl receptor appears to be an
important target of this pathway in embryonic (this study) and
larval tracheal cells. Knockdown of dVHL elevates btl
transcription in embryonic placodes and tracheal branches, and
removal of a copy of the gene effectively suppresses dVHL
tracheal phenotypes. Reciprocally, overexpression of wild type btl
in embryonic tracheal cells can produce migration defects and
sinuous overgrowth, while expression of a constitutively active btl
chimera (btlλ) also leads to primary branch stunting and
duplication of secondary branches. Interestingly, pupal lethality
associated with tracheal-specific knockdown of dVHL is not
sensitive to the dose of btl, but is dependent on sima.
Thus the dVHL/sima pathway may have btl
independent effects on tracheal cells in later stages of
development (Mortimer, 2009).
In addition to sima and Btl/FGF pathway
mutants, dVHL also shows very strong genetic
interactions with alleles of the ago ubiquitin ligase
subunit. The interactions are consistent with the ability of ago
to modulate hypoxia sensitivity in the embryo, and suggest a
speculative model in which each ligase acts through its own target
— Sima or Trh — to regulate btl
transcription in tracheal cells. Given that the human orthologs of
dVHL and ago are significant tumor suppressor
genes, it is intriguing to consider whether their ability to
co-regulate tubular morphogenesis in the Drosophila
embryo is conserved in mammalian development and disease
(Mortimer, 2009).
Go to top
Aso, T., Yamazaki, K., Aigaki, T. and
Kitajima, S. (2000). Drosophila von
Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin
ligase activity. Biochem Biophys Res Commun 276: 355-361. PubMed
ID: 11006129
Abstract
Mutations of the von Hippel-Lindau (VHL) tumor suppressor
gene predispose individuals to a variety of human tumors,
including renal cell carcinoma, hemangioblastoma of the central
nervous system, and pheochromocytoma. This study reports the
identification and characterization of the Drosophila
homolog of VHL. The predicted amino acid sequence of Drosophila
VHL protein shows 29% identity and 44% similarity to that of human
VHL protein. Biochemical studies show that Drosophila
VHL protein binds to Elongins B and C directly, and via this
Elongin BC complex, associates with Cul-2 and Rbx1. Like human
VHL, Drosophila VHL complex containing Cul-2, Rbx1,
Elongins B and C, exhibits E3 ubiquitin ligase activity. In
addition, hypoxia-inducible factor (HIF)-1α is the
ubiquitination target of both human and Drosophila VHL
complexes (Aso, 2000).
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Reviews
Hsu, T. (2012). Complex cellular
functions of the von Hippel-Lindau tumor suppressor gene: insights
from model organisms. Oncogene 31: 2247-2257. PubMed ID: 