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Current perspectives on the causes of neural tube defects resulting from diabetic pregnancy.

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American Journal of Medical Genetics Part C (Semin. Med. Genet.) 135C:77 – 87 (2005)
Current Perspectives on the Causes of Neural
Tube Defects Resulting From Diabetic Pregnancy
Maternal diabetes increases the risk for neural tube, and other, structural defects. The mother may have either
type 1 or type 2 diabetes, but the diabetes must be existing at the earliest stages of pregnancy, during which
organogenesis occurs. Abnormally high glucose levels in maternal blood, which leads to increased glucose
transport to the embryo, is responsible for the teratogenic effects of maternal diabetes. Consequently, expression
of genes that control essential developmental processes is disturbed. In this review, some of the biochemical
pathways by which excess glucose metabolism disturbs neural tube formation are discussed. Research from
the author’s laboratory has shown that expression of Pax3, a gene required for neural tube closure, is
significantly reduced by maternal diabetes, and this is associated with significantly increased neural tube defects
(NTD). Pax3 encodes a transcription factor that has recently been shown to inhibit p53-dependent apoptosis. Evidence in support of this model, in which excess glucose metabolism inhibits expression of Pax3,
thereby derepressing p53-dependent apoptosis of neuroepithelium and leading to NTD will be discussed.
ß 2005 Wiley-Liss, Inc.
KEY WORDS: pregnancy; Pax3; oxidative stress
Diabetes mellitus is a disease that has
affected humans, at least since the time of
the ancient Greeks (who gave the disease
the name, which roughly translates
to, ‘‘copious production of honeyedurine’’). There are two major forms of
diabetes, type 1 (formerly called, insulin-dependent diabetes or juvenile diabetes), in which the insulin-producing
beta cells of the pancreas are destroyed by
an autoimmune process, and type 2
(formerly called, non-insulin-dependent diabetes or adult-onset diabetes),
in which insulin-dependent glucose
uptake, primarily by fat and muscle,
becomes impaired. In recent years, the
terms, ‘‘insulin-dependent’’ and ‘‘non-
Mary R. Loeken is a Research Investigator
in the Section on Developmental and Stem
Cell Biology at the Joslin Diabetes Center,
and an Assistant Professor in the Department
of Medicine, Harvard Medical School.
*Correspondence to: Mary R. Loeken,
Joslin Diabetes Center, Harvard Medical
School, Boston, MA 02215.
DOI 10.1002/ajmg.c.30056
ß 2005 Wiley-Liss, Inc.
insulin-dependent’’ have fallen out of
use because even in type 2 diabetes,
compensatory increased insulin production by pancreatic beta cells eventually
causes insulin production to ‘‘exhaust,’’
necessitating insulin treatment. Similarly, the terms, ‘‘juvenile’’ and ‘‘adultonset’’ diabetes are misleading, because
both types of diabetes can occur in either
the young or the old. Although type 1
diabetes has its onset primarily before the
age of 20, it can occur in the 5th, or later,
decade of life. More notably, type 2
diabetes, which, until recently, occurred
primarily after the age of 40, has recently
reached epidemic rates in children and
young adults, owing to increased obesity
and sedentary lifestyles [Mokdad et al.,
2001; Kaufman, 2002].
The major long-term consequence
of diabetes is that if post-prandial glucose
fails to be transported and stored in fat
and muscle, circulating glucose concentrations remain higher than normal, and
chronic hyperglycemic exposure eventually leads to tissue pathology, including
diabetic retinopathy, nephropathy, vasculopathy, and neuropathy. Until the
availability of insulin in the 1920s, these
sequelae were primarily relevant to type
2 diabetes, as individuals with type 1
diabetes, having almost complete loss of
insulin-producing beta cells by the time
the disease was diagnosed, usually died
within a few years of onset of the disease.
Once insulin became available, longterm survival of children and young
adults with type 1 diabetes became
possible, and pregnancy in women with
pre-existing diabetes became increasingly more common. Maternal diabetes
increased risk for miscarriage, toxemia,
infant respiratory distress, and perinatal
hypoglycemia, placing the lives of the
infant, mother, or both at risk [White,
1937; Gabbe, 1993]. However, as management of these complications
improved, congenital malformations
remained the most significant adverse
outcome of diabetic pregnancy [White,
1949]. The recognition that severity of
pre-existing diabetic complications such
as retinopathy and nephropathy were
correlated with poor outcome of pregnancy, including congenital malformations, led to the development of the
White’s Classification Index of diabetic
pregnancy [White, 1949]. As will be
discussed further below, chronic poor
control of diabetes as manifested in
diabetic complications could be an
indicator of the glycemic exposure of
the embryo, and therefore its risk for
maldevelopment; alternatively, there
may be genetic modifiers which increase
or decrease risk for long-term diabetic
complications, which could also modify
risk for diabetic teratogenesis.
There have been several clinical
studies suggesting that the incidence
and severity of diabetic pregnancyinduced malformations are correlated
with poor glycemic control [Miller
et al., 1981; Greene et al., 1989; Lucas
et al., 1989; Langer and Conway, 2000;
Schaefer-Graf et al., 2000]. Most of
There have been several clinical
studies suggesting that the
incidence and severity of
diabetic pregnancy-induced
malformations are correlated
with poor glycemic control.
these studies are based on increased
non-enzymatic glycation of hemoglobin
(either HbA1 or HbA1C) as an index of
poor glycemic control. Non-enzymatic
glycation is a slow, mass action-dependent process [Furth, 1997]. Because of
the non-enzymatic nature of the reaction, the accumulation of glycated
proteins is directly related to the substrate (glucose) availability, and the
stability of the protein. Thus, glycation
of hemoglobin, a relatively long-lived
circulating protein, serves as a marker for
glycemic exposure during the approximately 60 preceding days, although it
does not indicate when excursions from
euglycemia occurred, nor the relative
peaks and nadirs of glycemia. However,
diabetes can cause malformation of
organ systems that are induced at
different times within the first several
weeks of gestation [Lucas et al., 1989;
Becerra et al., 1990; Martinez-Frias,
1994], indicating that precise windows
of susceptibility to specific organ defects
may be difficult to identify using glycated hemoglobin.
One large, prospectively-designed,
multi-center study found that malformations were increased 2.5-fold in the
offspring of diabetic women whose
pregnancies were identified within 21
days of conception, whereas they were
increased 4.5-fold if pregnancies were
identified later than 21 days following
conception [Mills et al., 1988]. In the
early entry group, there was no significant increase in glycated hemoglobin in
diabetic pregnancies, leading to the
conclusion that diabetes per se, and
not glycemic control, increased the
risk for malformation. However, this
conclusion was met with some
skepticism, as 93% of diabetic subjects
had HbA1 values as much as 7 standard
deviations above the non-diabetic
mean, which was not statistically significant in this analysis. It should also be
noted that glycated hemoglobin may
not be an appropriate indicator of
glycemic control and risk for diabetic
pregnancy-induced defect, not just
because it is an indicator of cumulative
glycemic exposure, rather than acute
exposure at critical stages of organogenesis, but also because of the increased
blood volume and erythropoiesis during
pregnancy progressively dilutes previously synthesized hemoglobin. Thus,
during pregnancy, glycated hemoglobin may under-reflect prior glycemic
Perhaps the best evidence in support of the role of poor glycemic control
and risk for malformation came following the Diabetes Complications Control
Trial (DCCT), in which 1,441 subjects
with type 1 diabetes enrolled in the study
at 29 sites throughout the US and
Canada between 1983 and 1993. Subjects were randomly assigned to conventional (i.e., without sampling of blood
glucose and insulin injection at frequent
intervals to attain euglycemia) or intensive (i.e., with four or more daily
samplings of blood glucose and adjustment of insulin delivery to attain euglycemia) diabetes therapy. This study
showed that intensive therapy lowered
glycated hemoglobin levels and reduced
the incidence and rate of progression of
the traditional diabetic complications
such as retinopathy, nephropathy,
amputations, and cardiovascular disease
[Anonymous, 1995a,b,c]. During the
course of the study, 180 women either
sought to become, or became, pregnant.
Because of the suspected contribution of
poor glycemic control to congenital
malformations, women who were originally assigned to the conventional
therapy group were reassigned to the
intensive therapy group [Group, 1996].
Glycated hemoglobin was significantly
higher at conception in women previously assigned to the conventional
control group compared to the intensive
control group (8.1% vs. 7.4%), but did
not differ during gestation (6.6% in both
groups). Notably, nine congenital malformations were identified, eight of
which occurred in the pregnancies
previously assigned to conventional
therapy. The conclusions from this study
were that rigorous glycemic control
during organogenesis can reduce the
risk for congenital malformations, but
that aggressive control should be
initiated prior to conception.
Nevertheless, even in the 21st
century, there continue to be reports
that birth defects, particularly those
affecting the neural tube and the heart,
are significantly increased (up to 5-fold)
in diabetic pregnancies [Langer and
Conway, 2000; Suhonen et al., 2000;
Aberg et al., 2001; Loffredo et al., 2001;
Sheffield et al., 2002; Wren et al.,
2003]. The interpretation from these
studies is generally that availability of
pre-pregnancy counseling on the importance of glycemic control, prevention of unplanned pregnancies by
diabetic women, and patient compliance
with intensive insulin therapy, should be
improved. However, another consideration is that altered production of hormones that regulate glucose homeostasis
upon pregnancy necessitates modification of insulin administration, diet, and
exercise in order to maintain appropriate
glycemic control. It should also be
recognized that even intensive insulin
therapy does not bring the rate of any
diabetic complication to the nondiabetic rate, and so it may be necessary
to develop additional interventions, or
perhaps, more physiologic methods of
controlling diabetes, before congenital
malformations in diabetic pregnancy
are no greater than in non-diabetic
It should also be recognized that
even intensive insulin therapy
does not bring the rate of any
diabetic complication to the
non-diabetic rate, and so it may
be necessary to develop
additional interventions, or
perhaps, more physiologic
methods of controlling diabetes,
before congenital
malformations in diabetic
pregnancy are no greater than in
non-diabetic pregnancy.
Excess glucose transported to the
embryo is responsible for the adverse
effects of maternal diabetes to cause
The previous discussion indicated
that as standards of diabetic treatment
have improved, the incidence and severity of congenital malformations have
been reduced. This would argue that the
cause of malformations in the embryos
of diabetic mothers is not inherently
linked to the disease (e.g., it is not
genetic or autoimmune in origin) but is
linked to the metabolic disruption of
normal fuel metabolism associated with
the disease. In considering the metabolic
disturbances of diabetes, which could be
responsible for congenital malformations, excess circulating glucose, insulin
(or insulin insufficiency), or other metabolic disturbances which are secondary
to defective glucose storage, such as
ketosis, altered circulating levels of
somatomedin-binding proteins, or
serum lipids, have been proposed. Circulating levels of ketones, somatomedin-binding proteins, and triglycerides
differ between type 1 and type 2
diabetics, and yet susceptibility to the
same kinds of congenital malformations
is the same in pregnancies affected by
either type 1 or type 2 diabetes [Towner
et al., 1995; Schaefer-Graf et al., 2000;
Aberg et al., 2001], although elevated
ketones may exacerbate the effects of
elevated glucose [Buchanan et al., 1994].
Insulin effects on the embryo (or effects
of insulin insufficiency) is not likely to be
responsible because maternal insulin is
not transported to the conceptus [Moore
and Persaud, 1993], and the period of
embryogenesis in which malformations
occur precedes the onset of fetal insulin
synthesis [Slack, 1995]. Circulating glucose, on the other hand, equilibrates
with the embryo [Sussman and
Matschinsky, 1988]. Despite other
metabolic differences, hyperglycemia is
a common characteristic of both type 1
or type 2 diabetes, supporting elevated
glucose as primarily responsible for malformations. Finally, post-implantation
culture of rat embryos in elevated
glucose-containing media induces more
numerous and more severe malformations than when cultured in low glucosecontaining media [Eriksson et al., 1982;
Eriksson, 1991]. However, the concentrations of glucose required to significantly increase malformations was far in
excess of those, which occur during
diabetes and increase risk for malformations, suggesting that glucose is not
primarily responsible for malformations,
or that the rat embryo culture is not a
sensitive model for glucose teratogenicity.
It is important to establish whether
glucose, or some other metabolic disturbances, are responsible for the adverse
effects of diabetic pregnancy on embryogenesis in order to investigate the
biochemical pathways involved.
It is important to establish
whether glucose, or some other
metabolic disturbances, are
responsible for the adverse
effects of diabetic pregnancy on
embryogenesis in order to
investigate the biochemical
pathways involved.
As will be explained further below,
we developed a mouse model of diabetic
pregnancy and showed that increased
neural tube defects (NTD) were correlated with reduced expression of Pax3, a
gene required for neural tube closure
[Phelan et al., 1997]. Using three
approaches, we showed that excess
glucose was responsible for decreased
Pax3 expression and increased NTD
[Fine et al., 1999]. First, embryo tissue
fragments were cultured in media containing 5 mM glucose (90 mg/dl) or
15 mM glucose (270 mg/dl). High
glucose inhibited Pax3 expression compared to culture in media containing
‘‘physiological’’ glucose; this was not
due to an osmotic effect of high glucose,
because excess L-glucose failed to inhibit
Pax3 expression. Second, inducing
hyperglycemia (250 mg/dl) in pregnant mice by glucose injection on the
day prior to the onset of Pax3 expression
significantly inhibited its expression and
increased NTD. Third, administration
of phlorizin, a drug which inhibits renal
tubular reabsorption of glucose, reduced
blood glucose levels of pregnant diabetic
mice, and the rate of NTD. These results
indicated that excess glucose delivered to
the embryo, as a consequence of maternal hyperglycemia, is necessary and
sufficient to cause malformations.
Furthermore, the fact that high glucose
inhibited Pax3 expression in embryo
tissue fragments suggested that the
effects of excess glucose were due to
direct effects on the embryo, and were
not due to, for example, a hostile uterine
environment, or even effects on extraembryonic structures.
The recognition that excess glucose, even episodic and transient, can be
teratogenic, may explain the increase
in malformations, especially NTD, in
pregnancies of obese women [Shaw
et al., 1996; Werler et al., 1996;
Moore et al., 2000; Mikhail et al.,
2002; Watkins et al., 2003]. Obese individuals may be insulin resistant, and so,
circulating glucose concentrations may
fluctuate above those of non-diabetic,
insulin sensitive individuals, or such
individuals may have undiagnosed type
2 diabetes. With the epidemic of obesity
among young adults and adolescents
(1; 2), the risk for birth defects and the
role of increased glucose exposure is an
important area of future investigation.
Virtually any organ system can be
affected by diabetic embryopathy, but
defects of the neural tube and the heart,
especially outflow tract defects, are
among the most frequently observed in
clinical studies [Becerra et al., 1990;
Martinez-Frias, 1994]. The NTD are
generally closure defects, especially spina
bifida, although non-closure defects,
such as holoprosencephaly or syntelencephaly are also observed [Barr et al.,
1983; Robin et al., 1996]. In rat embryos
from diabetic dams or cultured in high
glucose-containing media, externallydetectable malformations, including
exencephaly, spina bifida, micrognathia,
torsional defects, and growth and developmental retardation, have been
observed [Eriksson et al., 1982, 1991].
In mouse embryos of diabetic or glucose-injected dams, open NTD, especially exencephaly, are significantly
increased [Phelan et al., 1997; Fine
et al., 1999], although closed defects
resembling holoprosencephaly may also
occur [Phelan et al., 1997; Liao et al.,
2004]. Unlike the rat, there is no growth
or developmental delay observed in
mouse embryos of diabetic dams [Phelan
et al., 1997]. The morphological similarity and significant increase in NTD in
both rodent and human embryos by
diabetic pregnancy supports the validity
of rodent models to study the fundamental mechanisms by which these
defects occur.
Whereas most diabetes in humans is
a naturally-occurring disease resulting
from a combination of genetic susceptibility and environment, most rodent
studies of diabetic pregnancy have not
been performed using strains that naturally develop diabetes, but with strains in
which insulin-deficient diabetes is
induced with streptozotocin or alloxan,
both of which have primary effects to
destroy the b cells of the pancreas. There
are rodent models of type 2 diabetes, the
ob/ob and db/db mouse and the fa/fa
rat, all of which have defects in production or signaling of the anorexigenic
hormone, leptin [Beck, 2000]. How-
ever, since leptin is also required for
reproductive function in females
[Schubring et al., 2000], homozygous
animals are sterile and cannot be used for
the study of diabetic pregnancy. Perhaps
the best studied rodent model of type 1
diabetes, the non-obese diabetic (NOD)
mouse, develops diabetes with high
frequency (approximately 80% occurrence among females in the Joslin
colony), however, reproductive performance diminishes markedly once animals become manifestly diabetic.
(Nevertheless, severe NTD, including
exencephaly and craniorachischisis, was
observed in the fetuses of a diabetic
NOD mouse, which had successfully
delivered normal sized, non-malformed
litters prior to developing diabetes
[Phelan et al., 1997], suggesting that
increased malformations may occur as a
result of maternal diabetes in NOD
mice, and contribute to diminished
reproductive performance.)
New mouse models of diabetes,
with modifications of immune regulation
or insulin signaling are being generated
by transgenic and knockout technology
and may be useful for the study of
diabetic pregnancy-induced malformations. Nevertheless, having demonstrated
that maternal hyperglycemia is responsible for the adverse effects of diabetic
pregnancy on embryonic development, it
may be perfectly appropriate to employ
chemically induced models of insulin
insufficiency. In addition, a non-diabetic
animal can be used to perturb pathways,
which mediate the effects of high glucose
on embryogenesis to elucidate their
involvement in this process.
We developed a mouse model of
diabetic pregnancy in which diabetes is
induced about 3 weeks prior to pregnancy with streptozotocin. Streptozotocin has a half-life of 15 min [Gilman
et al., 1980], and its effects on embryogenesis can be prevented by sufficient
normalization of glucose in diabetic
animals [Fine et al., 1999], suggesting
that streptozotocin is not teratogenic per
se, when administered weeks prior to
organogenesis, but its adverse effects are
due to its induction of diabetic hyperglycemia. Within a week of streptozotocin administration, mice become
hyperglycemic (blood glucose levels
>300 mg/dl, compared to normal blood
glucose 110–150 mg/dl). Diabetes is
treated with subcutaneously implanted
insulin pellets that constitutively produce insulin. The insulin pellets control
the diabetes well enough that the mice
gain weight and ovulate normally. However, once mice become pregnant,
increased glucocorticoid production
and gluconeogenesis induce hyperglycemia (mean blood glucose >300 mg/
dl) beginning on day 4.5 of pregnancy.
Age-matched non-diabetic mice, however, have normal beta cell function and
maintain euglycemia during pregnancy
[Phelan et al., 1997]. Notably, in
embryos examined on day 10.5 or
11.5, NTD, primarily exencephaly, were
increased 3-fold compared to embryos
of non-diabetic mice [Phelan et al.,
1997]. The exencephaly was morphologically similar to those caused by
homozygous Splotch (Sp) alleles, which
result from null mutation of the Pax3
gene [Auerbach, 1954; Epstein et al.,
1993; Chalepakis et al., 1994]. We
showed that Pax3 expression is significantly inhibited in embryos of diabetic
mice beginning on day 8.5 of development [Phelan et al., 1997]. This is not
due to universal inhibition of gene
expression or developmental delay, as
expression of control genes such as
fibronectin, 36B4, and GAPDH was
not affected, and embryo growth and
development on day 8.5 (as determined
by crown/tail length, number of
somites, initiation of neural tube fusion)
was morphologically normal. This suggested that deficient expression of Pax3
in embryos of diabetic mothers phenocopied the effect of Pax3 loss of
Undoubtedly, there are other developmental control genes whose expression, depending on the timing and
severity of the diabetic state, could be
inhibited by maternal diabetes and lead
to embryonic defects. However, since
there are no redundant pathways to
compensate for Pax3 deficiency on
neural tube closure, simply reducing
Pax3 expression below a critical threshold will lead to a NTD with certainty.
Therefore, investigating how maternal
hyperglycemia disturbs the normal
expression of this gene, and how Pax3
deficiency leads to NTD, is important to
understand and prevent serious maldevelopment during diabetic pregnancy.
Molecular Regulation of Pax3 by
Excess Glucose in the Embryo
There is much evidence that reactive oxygen species (ROS) are increased
in tissues exposed to hyperglycemia, and
that oxidative stress plays a critical role in
the etiology of several diabetic complications [Giugliano and Ceriello, 1996;
Feldman et al., 1997; Ruggiero et al.,
1997; McDonagh and Hokama, 2000;
Vinik et al., 2000; Kowluru and
Kennedy, 2001]. In rodent embryos,
There is much evidence that
reactive oxygen species (ROS)
are increased in tissues exposed
to hyperglycemia, and that
oxidative stress plays a critical
role in the etiology of several
diabetic complications. In
rodent embryos, many studies
have shown that ROS are
increased by maternal diabetes
or high glucose culture, and that
administration of antioxidants,
or transgenic over expression of
Cuþþ/Znþþ superoxide
dismutase (SOD), can prevent
developmental defects.
many studies have shown that ROS are
increased by maternal diabetes or high
glucose culture, and that administration of antioxidants, or transgenic over
expression of Cuþþ/Znþþ superoxide
dismutase (SOD), can prevent hyperglycemia-induced developmental defects
[Eriksson and Borg, 1991; Hagay et al.,
1995; Eriksson and Siman, 1996; Sivan
et al., 1996, 1997; Wentzel et al., 1997;
Reece et al., 1998]. However, how
oxidative stress disturbs embryogenesis
has, until recently, been an unanswered
The distinct anatomical and developmental characteristics of the early
post-implantation embryo confer particular vulnerability to oxidative stress.
First of all, at this stage of development,
embryos express the high Km Glut2
glucose transporter (as well as the low
Km transporters, Glut1, Glut3, and
possibly, Glut8 [Hogan et al., 1991;
Takao et al., 1993; Carayannopoulos
et al., 2000], which may not be of
consequence during non-diabetic pregnancy, but could explain why intracellular glucose concentrations of the
embryo are at equilibrium with maternal
serum, regardless of maternal glycemia
[Sussman and Matschinsky, 1988]. Thus,
glucose flux into the embryo could be
2–3 (or more) times greater than during
non-diabetic pregnancy. Second, during
normal development, the early postimplantation embryo develops in a
relatively hypoxic environment (2%–
8% O2, compared to 20% in maternal
arterial circulation [Rodesch et al.,
1992; Fischer and Bavister, 1993]. This
is due to the absence of vascular tissue in
the embryo proper, and the several tissue
layers across which maternal O2 must
diffuse before reaching the embryo.
Since there is very little O2 available,
only about 8% of glucose is metabolized
oxidatively before establishment of the
embryo’s circulatory system [Akazawa
et al., 1994], and so, very little O
would be produced by the embryo
during normal development. Thus, it
makes physiologic sense that the embryo
does not express free radical scavenging
enzymes at high levels until shortly
before birth [Frank and Groseclose,
1984; el-Hage and Singh, 1990]. However, this being the case, even modest
increases in O
2 production could profoundly increase oxidative stress.
We hypothesized that pathways
activated by high glucose uptake by the
embryo would induce oxidative stress,
and that oxidative stress inhibits Pax3
expression. To test this, the diets of
diabetic and non-diabetic mice were
supplemented with vitamin E succinate,
raising the dietary intake of vitamin E
approximately 60-fold, and Pax3
mRNA and NTD were assayed [Chang
et al., 2003]. Vitamin E succinate
prevented the increase in malondialdehyde (a marker of lipid peroxidation,
used as an indicator of oxidative stress)
caused by maternal diabetes, without
affecting blood glucose concentrations;
vitamin E prevented the approximately
8-fold decrease in Pax3 mRNA in
embryos of diabetic mice, as well as the
significant increase in NTD.
Conversely, to test whether
increased oxidative stress inhibits Pax3
expression, pregnant mice were injected
with antimycin A (AA), an inhibitor of
complex III of electron transport, to
increase O2 production [Turrens et al.,
1985; Garcia-Ruiz et al., 1995]. AA
(which had no effect on blood glucose
levels) significantly inhibited Pax3
expression and increased NTD, showing
that oxidative stress is sufficient to elicit
these effects of maternal diabetes.
As an additional approach, the
effects of antioxidants to block the effects
of high glucose-containing media on
Pax3 expression was tested. Vitamin E,
and also the membrane-permeable analog of the important cellular antioxidant,
reduced glutathione (GSH), GSH ethyl
ester, prevented the inhibition of Pax3
expression, and AA replicated the effects
of high glucose, demonstrating that the
adverse effects of glucose to increase
oxidative stress occurred within the
embryo. The effect of oxidative
stress to inhibit Pax3 expression was
not due to direct cytotoxicity resulting
in death of Pax3-expressing cells, as AA
which is a much more potent oxidant
than glucose metabolism, did not induce
DNA strand breaks at concentrations
that were sufficient to inhibit Pax3
Potential Mechanisms by Which
Oxidative Stress Inhibits
Pax3 Expression
While it is not known how oxidative stress could affect embryo gene
expression, there are examples from
other systems. For example, in the NF-
kB pathway, oxidative stress can activate
I-kB kinase (IKK) which phosphorylates IkB, which thereby dissociates from
NF-kB, allowing NF-kB to translocate
to the nucleus and regulate gene expression [Karin and Ben-Neriah, 2000].
There are many other examples in which
transcriptional activities or DNA binding
of factors, such as p53, AP-1, and Sp1, are
regulated by redox status [Cox et al.,
1995; Wu et al., 1996; Jayaraman
et al., 1997; Marshall et al., 2000; Moran
et al., 2001; Webster et al., 2001].
In addition to mitochondrial production of O
2 , there are several metabolic pathways that regulate redox status
and may have transcriptional effects. For
example, increased glycolytic flux stimulates the hexosamine biosynthetic
pathway (HBP), in which the enzyme,
glutamine:fructose-6-phosphate amidotransferase (GFAT), catalyzes the
amido transfer from glutamine to the
glycolytic intermediate, fructose-6phosphate, yielding glucosamine-6phosphate [Marshall et al., 1991]. The
HBP provides the substrates for Olinked glycosylation of proteins therefore, increased HBP flux could increase
glycosylation, rather than phosphorylation, of cytoplasmic or nuclear proteins
[Wells et al., 2001]. Glucosamine-6phosphate also inhibits glucose-6-phosphate dehydrogenase (G6PD), the ratelimiting enzyme of the pentose shunt
pathway [Kanji et al., 1976]. G6PD
activity is coupled to reduction of
important regulator of redox status by
serving as a co-factor for glutathione
reductase, which converts oxidized glutathione (GSSG) to reduced glutathione
(GSH), and by enhancing the activity of
catalase, which converts H2O2 to H2O
and O2. The ratios of oxidized and
reduced NAD co-factors have been
shown to regulate transcription factor
binding and expression of circadian
clock genes [Rutter et al., 2002]. In
addition, cell cycle-dependent activation of histone H2B transcription
requires the glycolytic enzyme, glyceraldehyde-3-PO4 (GAPDH), as a coactivator, and that association of
GAPDH with a DNA-binding complex
is enhanced by NAD and inhibited by
NADH [Zheng et al., 2003]. GAPDH
activity may be inhibited by mitochondrial production of O
2 [Nishikawa
et al., 2000a,b] which would increase
NAD:NADH. Inhibition of GAPDH
activity is linked to malformations in rat
embryos [Wentzel et al., 2003], although
it is not known whether this is due to
altered ratios of NAD co-factors or
transcriptional effects of GAPDH, or
an indirect effect of inhibition of
GAPDH activity. Inhibition of GAPDH
may increase accumulation of diacylglycerol (DAG) and stimulation of protein
kinase C (PKC) [Nishikawa et al.,
2000a,b]. We have observed that DAG
and PKC are stimulated in embryos of
diabetic and glucose-injected mice [Hiramatsu et al., 2002], although we do not
yet know whether increased PKC activity is mechanistically involved in altered
gene expression and malformations.
While activation of PKC could directly
regulate transcription, PKC also stimulates NADPH oxidase activity [Hua
et al., 2003], which would contribute
to decreased production of GSH.
Clearly, there are multiple interacting
biochemical processes by which
increased glucose metabolism in the
embryo can increase oxidant production
and inhibit scavenging. Precisely how
oxidative stress interferes with developmental control of transcription in the
embryo will depend upon identification
of the transcription factors involved,
and understanding how their activation
during development is disturbed by
increased oxidant status.
Susceptibility to Inhibition of
Pax3 Expression by Maternal
Diabetes may be Modified
by Genetic Background
In humans, there is evidence that
genetic background and ethnicity influence susceptibility to diabetic complications such as nephropathy and
cardiovascular disease, independent of
glycemic control [Doria et al., 1995;
Cappuccio, 1997; Weijers et al., 1997;
Hosey et al., 1998; Martins et al., 2002].
While it is known that ethnicity is a
factor in the development of gestational
diabetes [Homko et al., 1995], which
While it is known that
ethnicity is a factor in the
development of gestational
diabetes, which increases risk
for subsequent pre-gestational
type 2 diabetes, the contribution
of genetic background to
diabetic embryopathy in
humans has not been carefully
examined. In animal models,
penetrance or expressivity of a
phenotype associated with a
genetic mutation is well known
to be influenced by strain
background, although the
specific modifier gene(s)
responsible are seldom
increases risk for subsequent pregestational type 2 diabetes [SchaeferGraf et al., 2002] the contribution of
genetic background to diabetic embryopathy in humans has not been carefully
examined. In animal models, penetrance
or expressivity of a phenotype associated
with a genetic mutation is well known to
be influenced by strain background
[Linder, 2001], although the specific
modifier gene(s) responsible are seldom
characterized. In the rat, the effects of
maternal diabetes to induce malformations is strain dependent [Eriksson,
1988]. Embryos from a malformationresistant rat strain express increased levels
of Mnþþ-SOD and catalase mRNA,
suggesting that higher levels of free
radical scavenging enzymes protect these
embryos from oxidative stress caused by
maternal diabetes [Cederberg et al.,
We have found one mouse strain,
C57Bl/6J, in which diabetic pregnancy
fails to increase NTD, whereas diabetic
pregnancy generally increases NTD
three or more fold in several other strains
(FVB, ICR, 129/Sv) [Pani et al., 2002a].
We hypothesized that in C57Bl/6J
embryos, maternal diabetes fails to
inhibit Pax3 expression below a critical
threshold. If this hypothesis is correct,
then investigating the genetic basis for
resistance to this effect of maternal
diabetes could point to essential biochemical pathways involved. Furthermore, if susceptibility to diabetic
pregnancy-induced NTD is polymorphic in humans as well as in mice,
it may be possible to screen individuals
for risk for this cause of NTD.
Consistent with the hypothesis,
maternal diabetes significantly inhibits
Pax3 expression in a strain that is
susceptible to diabetes-induced NTD,
FVB, but not in C57Bl/6J embryos.
Resistance to NTD caused by diabetic
pregnancy is determined by the genotype of the embryo and is a dominant
trait, as heterozygous C57-FVB
embryos are resistant whether the
mother or the father is of the resistant
strain. There is no significant difference
between strains in expression of catalase,
Cuþþ/Znþþ SOD, Mnþþ-SOD, gglutamcystein synthetase, and glutathione peroxidase, suggesting that
expression of genes that regulated free
radical scavenging does not account for
the genetic differences. However, differences in enzymatic activity, rate of free
radical generation, or other glucoseregulated pathways are some possible
explanations for the difference between
susceptible and resistant strains.
Insufficient Expression of Pax3
Leads to NTD by De-Repressing
p53-Dependent Apoptosis
Pax3 encodes a DNA-binding transcription factor [Goulding et al., 1991],
and several groups have found that
expression of a number of genes (those
encoding N-CAM, myelin basic protein,
the c-Met receptor, MyoD and Myf-5,
Pax7, and Msx2) is up- or downregulated by Pax3 [Laborda, 1991;
Moase and Trasler, 1991; Kioussi et al.,
1995; Epstein et al., 1996; Maroto et al.,
1997; Tajbakhsh et al., 1997; Borycki
et al., 1999; Kwang et al., 2002]. In
addition, we identified two additional
genes, Dep-1, and cdc46, whose expres-
sion is increased and decreased, respectively, by Pax3 [Cai et al., 1998; Hill
et al., 1998]. Therefore, it has generally
been held that Pax3 participates in the
regulation of genes that direct tissuespecific morphogenesis. However, of
the genes for which transcription control elements have been characterized,
only MyoD contains an element with
optimal binding sites for both the paired
and homeodomains of Pax3 which we
identified to be essential for high affinity
binding [Phelan and Loeken, 1998].
This raises the question of whether
altered expression of at least some of
these genes may be indirect, or secondary to some other Pax3-dependent
process. Expression of Pax3, as well as
other members of the Pax family, had
been shown to cause oncogenic transformation of cell lines and tumor
formation in nude mice [Maulbecker
and Gruss, 1993], and translocation of a
part of the human PAX3 gene encoding
the DNA binding domains is associated
with pediatric rhabdomyosarcoma [Barr
et al., 1993]. Furthermore, down regulation of the protein produced by this
translocation with PAX3 antisense oligonucleotides activates cell death of
rhabdomyosarcoma cells in culture [Bernasconi et al., 1996]. This prompted us
to investigate whether Pax3 might be
necessary for cell viability during neural
tube formation. We found that Pax3
deficiency, caused either by reduced
expression from maternal diabetes or
the Sp/Sp genotype, leads to apoptosis.
This, then, provided a cellular explanation for NTD resulting from Pax3
deficiency [Phelan et al., 1997]. Our
studies were limited to the neural tube,
but others showed that Pax3 is necessary
for migration of neural crest cells and
limb muscle precursors [Daston et al.,
1996; Yang et al., 1996; Conway et al.,
1997] and that apoptosis is prevalent in
newly formed somites of Sp/Sp embryo
[Borycki et al., 1999]. Therefore, loss of
Pax3 expressing cells by apoptosis
appears to be a general feature of
abnormal development in Pax3 deficient embryos. However, it was not clear
if Pax3 inhibits a cell death program, or if
apoptosis resulted from failure of a Pax3dependent developmental program.
To examine the regulation of apoptosis by Pax3 more closely, and specifically, to test whether this apoptosis occurs
by a p53-dependent mechanism, we
crossed Sp/þ and p53þ/ mice to
generate double heterozygotes [Pani
et al., 2002b]. Sp/þ p53þ/ mice were
crossed, and embryos were obtained on
day 10.5. As an additional approach, a p53
inhibitor, pifithrin-a, was administered
during neural tube formation (days 8.5
and 9.5) to pregnant Sp/þ mice. As
expected, all Sp/Sp embryos that were
w.t. at the p53 locus developed spina
bifida exencephaly with 100% penetrance, and TUNEL-positive cells were
abundant at sites of NTD. Remarkably,
homozygous p53 deficiency rescued not
only apoptosis in Sp/Sp embryos, but also
neural tube closure, as none of the Sp/Sp
p53/ embryos displayed NTD. p53
heterozygosity or inhibition with pifithrin-a prevented NTD in about half of
the Sp/Sp embryos. These results profoundly alter our understanding of how
Pax3 directs neural tube closure. Since
neural tube closure was completely
normal in Sp/Sp embryos as long as they
were p53-deficient, this suggests that
neural tube closure is a Pax3-independent
process, and the sole required function of
Pax3 in neural tube closure is to inhibit
p53-dependent apoptosis.
Since Pax3 is a transcription factor,
and there is evidence that another
member of the Pax family, Pax5, inhibits
p53 expression at the transcriptional
level [Stuart et al., 1995], we considered that Pax3 could inhibit p53 gene
expression. On the other hand, acute
regulation of p53 protein levels is
primarily post-translational, due to dissociation from mdm2, a ubiquitin ligase
that stimulates proteosome-dependent
degradation [Colman et al., 2000].
Phosphorylation or acetylation of p53
in response to stresses, such as radiation,
can inhibit association with mdm2, and
enhance p53 stability [Craig et al., 1999].
Activation of PKB/Akt increases phosphorylation of mdm2, which enhances it
association with p53, thereby decreasing
p53 stability, but the PTEN tumor
suppressor protein inhibits activation of
Akt, thereby enhancing p53 stability
[Mayo and Donner, 2002]. P14(Arf)
binds to mdm2 and sequesters it away
from p53, thereby enhancing p53 stability [Kamijo et al., 1998]. There is also
evidence that p53 protein levels may
regulated at the level of translation, as
there are sequences that suppress translation of p53 in response to UV or g
irradiation or growth arrest that are
located in both the 50 and 30 UTR’s
[Mosner et al., 1995; Fu et al., 1999]. A
40 kDa COO-terminal fragment of p53
may derepress the 50 UTR [MokdadGargouri et al., 2001], while the RNA
binding protein, HuR, specifically binds
to the 30 UTR of p53 mRNA and
enhances p53 translation in response to
UV irradiation [Mazan-Mamczarz et al.,
To test whether the inhibition of
p53-dependent apoptosis by Pax3 might
be due to either inhibition of p53
mRNA or protein accumulation, we
assayed p53 mRNA levels in w.t., Sp/þ,
and Sp/Sp embryos by RT-PCR, and
p53 protein levels by immunoblot.
There was no effect of Pax3 on p53
mRNA. However, in whole embryos,
there was about 2-fold less p53 protein in
w.t. compared to Sp/Sp embryos.
Because these assays were performed
using whole embryos, and Pax3 is
expressed at only limited sites in the
embryo, the magnitude of the effect of
Pax3 on p53 in Pax3-expressing cells is
probably much more than 2-fold.
Understanding exactly how Pax3 regulates p53 either translationally or posttranslationally will require additional
A related question is why Pax3 is
required to inhibit p53-dependent
apoptosis. Noting that transformation
of fibroblasts with Pax-expressing plasmids induces focus formation in vitro
and tumors in nude mice [Maulbecker
and Gruss, 1993], and that PAX3 is
expressed in several human neuroepithelial, neural crest, or myoblastderived malignancies such a melanoma,
neuroblastoma, pediatric rhabdomyosarcoma, and Ewing’s sarcoma [Galili
et al., 1993; Shapiro et al., 1993; Schulte
et al., 1997; Barr et al., 1999; Vachtenheim and Novotna, 1999; Scholl et al.,
2001; Harris et al., 2002], it is possible
that p53 activates cell cycle withdrawal.
Repression of this could be essential for
the growth and migration of neural crest
and neuroepithelium as well as proper
dorsal ventral patterning of the neural
tube. However, when PAX3 is inappropriately expressed in tumors, repression
of p53-dependent cell cycle withdrawal
may contribute to tumor growth. Alternatively, noting that oxidative stress can
activate p53 and lead to cell death
[Webster and Perkins, 1999; Trinei
et al., 2002; Menendez et al., 2003],
and that the sudden increase in delivery
in oxygen to the embryo upon the
establishment of its own cardiovascular
system and blood supply, which occurs
more or less coincident with the beginning of neural tube closure, could
increase O
2 and activation cell death, it
may be necessary to activate expression
of genes which would override p53induced cell death. Thus, during
diabetic pregnancy, the increased neuroepithelial apoptosis leading to NTD
may be due to a combination of oxidative stress-induced stabilization of p53,
along with de-repression of p53 due to
insufficient expression of Pax3.
A general scheme to explain how
diabetic pregnancy causes NTD can be
summarized as follows:
1. Maternal diabetes increases glucose
concentrations in maternal circulation, including in the uterine artery.
2. Glucose, originating from the maternal circulation, is freely transported
to the embryo and transported into
embryo cells.
3. Increased glucose taken up by
embryo cells, through a combination
of increased oxidative metabolism at a
stage of development in which free
radical scavenging is immature, and
altered activities of biochemical pathways that contribute to redox homeostasis, causes oxidative stress in the
4. Oxidative stress leads to decreased
expression of Pax3, a gene required
for neuroepithelial and neural crest
development. Expression of other
genes that participate in the forma-
tion of the neural tube, or other
organs, may also be affected, but since
there are no redundant pathways,
which compensate for Pax3 deficiency, simply reducing the expression of Pax3 below a critical threshold
at vulnerable stages in neural tube
formation may be sufficient to
induce a NTD.
5. As a result of decreased production of
Pax3, synthesis or stability of p53
protein increases, and this activates
cell death, which aborts the process of
neural tube closure.
It should be noted that deficient
Pax3 expression leading to derepression
of p53-dependent apoptosis may be
involved in NTD resulting from causes
other than diabetic pregnancy. For
example, ionizing radiation and certain
anticonvulsant drugs may induce oxidative stress [Nicol et al., 2000; Agrawal
et al., 2001]. While the mechanisms by
which increased folic acid administration reduces NTD in genetically susceptible individuals is not well understood,
folate increases the production of
methionine from homocysteine, thereby preventing the accumulation of
homocysteine, which is an oxidant
[Rosenquist et al., 1996; Chern et al.,
2001]. In addition, folic acid may
have antioxidant properties of its own
[Nakano et al., 2001]. Thus, while it
remains to be experimentally demonstrated, deficient Pax3 expression may
result from multiple different sources of
increased oxidant status in the embryo at
critical stages of neural tube closure.
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