Pten
Inactivating mutations in the PTEN tumor suppressor gene, encoding a phosphatase, occur in three related human autosomal dominant disorders characterized by
tumor susceptibility. Pten heterozygous (Pten+/-) mutants develop a lethal polyclonal autoimmune disorder with features reminiscent of those
observed in Fas-deficient mutants. Fas-mediated apoptosis is impaired in Pten+/- mice, and T lymphocytes from these mice show reduced activation-induced cell
death and increased proliferation upon activation. Phosphatidylinositol (PI) 3-kinase inhibitors restore Fas responsiveness in Pten+/- cells. These results indicate
that Pten is an essential mediator of the Fas response and a repressor of autoimmunity and thus implicate the PI 3-kinase/Akt pathway in Fas-mediated apoptosis (Di Christofano, 1999).
A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. A missense mutation
in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a
substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or
substrate-trapping forms of PTEN in HEK293 cells alters the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the
phosphatase in PTEN-deficient tumor cell lines results in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival (Myers, 1998).
To investigate the molecular basis of PTEN-mediated tumor suppression, a null mutation was introduced into the mouse Pten gene by homologous recombination in
embryonic stem (ES) cells. Pten-/- ES cells exhibit an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also
display advanced entry into S phase. This accelerated G1/S transition is accompanied by down-regulation of p27(KIP1), a major inhibitor for G1
cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts results in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a
product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency leads to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase
B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increases Bad phosphorylation and promotes Pten-/- cell
survival. These studies suggest that PTEN regulates the phosphatidylinositol 3,4, 5,-trisphosphate and Akt signaling pathway and consequently modulates two critical
cellular processes: cell cycle progression and cell survival (Sun, 1999).
The mechanism by which PTEN mediates growth inhibition was investigated. Using the mutant PTEN glioma cell
line, U87MG, as a transfection recipient for a series of PTEN alleles, provide direct evidence is provided that the capacity for growth inhibition requires phosphatase activity. Mutations mapping
upstream, within, and downstream of the catalytic domain ablate activity toward a 3' phosphorylated phosphoinositide substrate of PTEN, whereas alleles with
mutations flanking the catalytic domain retains activity toward the acidic protein polymer substrate, Glu4Tyr1. Thus, catalytic activity toward phosphoinositide
substrates is required for growth suppression, whereas activity toward the protein substrate is dispensable for growth suppression. Finally, apoptotic
and cell proliferation analyses were used to show that PTEN-mediated growth inhibition under reduced serum conditions is due to a G1 cell cycle block rather than to an
induction of apoptosis (Di Cristofano, 1999).
PTEN expression potently suppresses the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN is
mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlates with a significant increase of the cell cycle kinase inhibitor
p27(KIP1) and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also leads to the inhibition of Akt/protein kinase B, a
serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27(KIP1) and the cell
cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, these studies suggest that the PTEN tumor
suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is
the cyclin-dependent kinase inhibitor p27(KIP1) (D. Li, 1998).
PTEN inhibited cell growth and/or colony formation in all of epithelial lines tested with one exception. The
decrease in cellular proliferation is associated with an induction of apoptosis and an inhibition of signaling through the phosphatidylinositol 3'-kinase pathway.
Akt/protein kinase B, a gene whose antiapoptotic function is regulated by phosphatidylinositol-3,4,5-triphosphate, is able to rescue cells from PTEN-dependent
death. PTEN, therefore, appears to suppress tumor growth by regulating phosphatidylinositol 3'-kinase signaling (J. Li, 1998).
The tumour suppressor PTEN, also named MMAC1 or TEP1, is associated with a number of malignancies in human populations. This protein has a dual protein
phosphatase activity, being also capable to dephosphorylate phosphatidylinositol 3,4,5 triphosphate. The mechanism of growth suppression
attributable to PTEN has been studied. PTEN overexpression inhibits cell growth in a variety of normal and transformed, human and murine cells.
Bromodeoxyuridine (BrdU) incorporation and TUNEL labelling experiments in transiently transfected cells demonstrate that this inhibition is due to a cell cycle arrest
rather than induction of apoptosis. Given that PTEN is unable to cause cell growth arrest in retinoblastoma (Rb)-deficient cell lines, the possible
requirement for pRb in the PTEN-induced inhibition of cell proliferation has been investigated. The co-expression of SV40 antigen, but not a mutant form (which binds
exclusively to p53), and cyclin D1/cdk4 are able to overcome the PTEN-mediated growth suppression. In addition, the reintroduction of a functional pRb, but not its
relatives p107 or p130, in Rb-deficient cells restores the sensitivity to PTEN-induced arrest. Finally, the hyperphosphorylation of transfected pRb is inhibited by
PTEN co-expression and restored by PI-3K co-expression. Accordingly, PTEN gene is mostly expressed, in parallel to Akt, in mid-late G1 phase during cell cycle
progression prior to pRb hyperphosphorylation. Finally, the signal transduction pathways modulated by PTEN expression have been investigated. PTEN-induced growth arrest can be rescued by the co-expression of active PI-3K and downstream effectors such as Akt or PDK1, and also certain small
GTPases such as Rac1 and Cdc42, but not by active Ha-ras, raf or RhoA. Collectively, these data link the tumour suppressor activities of PTEN to the machinery
controlling cell cycle through the modulation of signalling molecules whose final target is the functional inactivation of the retinoblastoma gene product (Paramio, 1999).
PTEN/MMAC1/TEP1, a tumor suppressor gene, is frequently mutated in a variety of human cancers. Germ-line mutations of phosphatase and tensin homolog,
deleted on chromosome ten (PTEN) are found in two inherited hamartoma tumor syndromes: Cowden syndrome, which has a high risk of breast, thyroid, and other
cancers; and Bannayan-Zonana syndrome, a related disorder. PTEN encodes a phosphatase that recognizes both protein substrates and
phosphatidylinositol-3,4,5-triphosphate. The lipid phosphatase activity of PTEN seems to be important for growth suppression through inhibition of the
phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Clones were established with stable PTEN expression controlled by a tetracycline-inducible system to
examine the consequences of increased levels of wild-type and mutant PTEN expression in a well-characterized breast cancer line, MCF-7. When PTEN is
overexpressed in MCF-7, growth suppression is observed, but only if PTEN phosphatase activity is preserved. The initial growth suppression is
attributable to G1 cell cycle arrest, whereas subsequent growth suppression is attributable to a combination of G1 arrest and cell death. Of note, the decrease in
Akt phosphorylation precedes the onset-of suppression of cell growth. Treatment of MCF-7 cells with wortmannin, a PI3K inhibitor, causes cell growth inhibition in
a way similar to the effects of overexpression of PTEN in this cell. In general, the inverse correlation between PTEN protein level and Akt phosphorylation is
found in a panel of breast cancer cell lines. Therefore, PTEN appears to suppress breast cancer growth through down-regulating PI3K signaling, which leads to the
blockage of cell cycle progression and the induction of cell death, in a sequential manner (Weng, 1999).
The PTEN tumor suppressor acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3). PTEN negatively controls the G1/S cell cycle transition
and regulates the levels of p27KIP1, a CDK inhibitor. Ubiquitin E3 ligase is an SCFSKP2 complex that mediates p27 ubiquitin-dependent proteolysis. PTEN and the PI 3-kinase pathway regulate p27 protein stability. PTEN-deficiency in mouse embryonic stem (ES) cells causes a decrease of p27 levels with concomitant increase of SKP2, a key component of the SCFSKP2 complex. Conversely, in human glioblastoma cells, ectopic
PTEN expression leads to p27 accumulation, which is accompanied by a reduction of SKP2. Ectopic expression of SKP2 alone is sufficient to reverse PTEN-induced p27 accumulation, restore the kinase activity of cyclin E/CDK2, and partially overcome the PTEN-induced G1 cell cycle arrest. Consistently, recombinant SCFSKP2 complex or SKP2 protein alone can rescue the defect in p27 ubiquitination in extracts prepared from cells treated with a PI 3-kinase inhibitor. These findings suggest that SKP2 functions as a critical component in the PTEN/PI 3-kinase pathway for the regulation of p27KIP1 and cell proliferation (Mamillapalli, 2001).
Adhesion between epithelial cells mediates apical-basal polarization, cell proliferation, and survival, and defects in adhesion junctions are associated with abnormalities from degeneration to cancer. The maintenance of specialized adhesions between cells of the retinal pigment epithelium (RPE) requires the phosphatase PTEN. RPE-specific deletion of the mouse pten gene results in RPE cells that fail to maintain basolateral adhesions, undergo an epithelial-to-mesenchymal transition (EMT), and subsequently migrate out of the retina entirely. These events in turn lead to the progressive death of photoreceptors. The C-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding domain of PTEN is essential for the maintenance of RPE cell junctional integrity. Inactivation of PTEN, and loss of its interaction with junctional proteins, are also evident in RPE cells isolated from CC chemokine receptor 2-deficient ccr2-/- mice and from mice subjected to oxidative damage, both of which display age-related macular degeneration (AMD). Together, these results highlight an essential role for PTEN in normal RPE cell function and in the response of these cells to oxidative stress (Kim, 2008).
To examine the role of PTEN in ontogenesis and tumour suppression, mouse Pten was disrupted by homologous
recombination. Pten inactivation results in early embryonic lethality. Pten-/- ES cells form aberrant embryoid bodies and display an altered ability to
differentiate into endodermal, ectodermal and mesodermal derivatives. Pten+/- mice and chimaeric mice derived from Pten+/- ES cells show
hyperplastic-dysplastic changes in the prostate, skin and colon, which are characteristic of CD, LDD and BZS. They also spontaneously developed germ cell,
gonadostromal, thyroid and colon tumours. In addition, Pten inactivation enhances the ability of ES cells to generate tumours in nude and syngeneic mice, due to
increased anchorage-independent growth and aberrant differentiation. These results support the notion that PTEN haploinsufficiency plays a causal role in tumorogenesis, and demonstrate that Pten is a tumour suppressor essential for embryonic development (Di Cristofano, 1998).
Homozygous mutant mice lacking exons 3-5 of the PTEN gene (mPTEN3-5) have severely expanded and abnormally patterned
cephalic and caudal regions at day 8.5 of gestation. Embryonic death occurs by day 9.5 and is associated with defective chorio-allantoic development.
Heterozygous mPTEN3-5 mice have an increased incidence of tumors, especially T-cell lymphomas; gamma-irradiation reduces the time lapse of tumor formation.
DNA analysis of these tumors reveals the deletion of the mPTEN gene due to loss of heterozygosity of the wild-type allele. Tumors associated with loss of
heterozygosity in mPTEN show elevated phosphorylation of protein kinase B (PKB, also known as Akt kinase), thus providing a functional connection between
mPTEN and a murine proto-oncogene (c-Akt) involved in the development of lymphomas. It is concluded that the mPTEN gene is fundamental for embryonic
development in mice, as mPTEN3-5 mutant embryos die by day 9.5 of gestation, with patterning defects in cephalic and caudal regions and defective placentation.
Heterozygous mice developed lymphomas associated with loss of heterozygosity of the wild-type mPTEN allele, and tumor appearance is accelerated by
gamma-irradiation. These lymphomas have high levels of activated Akt/PKB, the protein product of a murine proto-oncogene with anti-apoptotic function, associated
with thymic lymphomas. This suggests that tumors associated with mPTEN loss of heterozygosity may arise as a consequence of an acquired survival advantage. Direct evidence of the role of mPTEN as a tumor suppressor gene in mice is provided, and the mPTEN mutant mouse is established as an experimental model for
investigating the role of PTEN in cancer progression (Suzuki, 1998).
The cerebellar cortex is a model system for studies on
glial-guided neuronal migration and on glial differentiation.
Bergmann glial cells are first seen in the cerebellar cortex in the
late embryonic period, when they express the markers RC2 and BLBP. By birth, the
radial glial population has disappeared, replaced by the Bergmann glia, which
extend processes from midway through the anlagen to the pial surface. The
migration of postmitotic-granule cell precursors begins after birth, with
maximal periods of migration between P7 and P12. By P15, the external granule
layer (EGL) is no longer evident, since the progenitors have all migrated into the
internal granule cell layer (IGL), a zone just deep to the Purkinje neurons.
Evidence is provided that loss of PTEN in Bergmann glia leads to premature differentiation and subsequently to extensive layering defects.
Accordingly, severe granule neuron migration defects and abnormal laminar
formation are observed. These results uncover an unexpected role for PTEN in
regulating Bergmann glia differentiation, as well as the importance of
time-dependent Bergmann glia differentiation during cerebellar development (Yue, 2005).
Evidence is provided that the interplay between Bergmann
glial scaffold and granule neurons is vital for granule neuron migration and
IGL formation. When selectively deleting Pten in Bergmann glia by
targeted adenovirus injection in vivo at P3, prior to the peak of granule
neuron migration (P6-7), the structural changes in
Pten-null Bergmann glia are sufficient to affect granule neuron
migration. However, no significant granule neuron migration
defect is observed after injecting adenovirus at P7, when granule neuron migration is still near its peak. Whereas biochemical changes occur as early as 12-24 hours
after Pten deletion, the structural changes require at least 3-4
days. These data argue that astrocytic structural changes of
Pten-null Bergmann glia are likely to be the primary cause of granule
neuron migration defects and that the effect of Bergmann glia on granule cell
migration is a developmental stage-dependent event. Furthermore, the data
suggest that Bergmann glial scaffold may be important for Purkinje cell
dendritic arborization. A recent study suggested that Purkinje cell dendritic
arborization may rely on Bergmann fibers for proper alignment.
Consistent with this, a time-dependent Purkinje dendritic defect was observed
in PTEN mutants, strictly coupled with the timecourse of Bergmann glia changes. As the Bergmann fibers disappear, the Purkinje dendrites become randomly oriented and show a lack of fine processes (Yue, 2005).
Loss of PTEN causes unregulated activation of downstream components of phosphatidylinositol 3-kinase (PI3K) signaling, including PDK1, and disrupts normal nervous system development and homeostasis. This study tested the contribution of Pdk1 to the abnormalities induced by Pten deletion in the brain. Conditional deletion of Pdk1 caused microcephaly. Combined deletion of Pdk1 and Pten rescued hypertrophy, but not migration defects of Pten-deficient neurons. Pdk1 inactivation induced strikingly different effects on the regulation of phosphorylated Akt in glia versus neurons. These results show Pdk1-dependent and Pdk1-independent abnormalities in Pten-deficient brains, and demonstrate cell type specific differences in feedback regulation of the ubiquitous PI3K pathway (Chalhoub, 2009).
The failure of axons to regenerate is a major obstacle for functional recovery after central nervous system (CNS) injury. Removing extracellular inhibitory molecules results in limited axon regeneration in vivo. To test for the role of intrinsic impediments to axon regrowth, cell growth control genes were analyzed using a virus-assisted in vivo conditional knockout approach. Deletion of PTEN (phosphatase and tensin homolog), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, in adult retinal ganglion cells (RGCs) promotes robust axon regeneration after optic nerve injury. In wild-type adult mice, the mTOR activity is suppressed and new protein synthesis is impaired in axotomized RGCs, which may contribute to the regeneration failure. Reactivating this pathway by conditional knockout of tuberous sclerosis complex 1, another negative regulator of the mTOR pathway, also leads to axon regeneration. Thus, these results suggest the manipulation of intrinsic growth control pathways as a therapeutic approach to promote axon regeneration after CNS injury (Park, 2008).
Despite the essential role of the corticospinal tract (CST) in controlling voluntary movements, successful regeneration of large numbers of injured CST axons beyond a spinal cord lesion has never been achieved. This study found that PTEN/mTOR are critical for controlling the regenerative capacity of mouse corticospinal neurons. After development, the regrowth potential of CST axons is lost and this is accompanied by a downregulation of mTOR activity in corticospinal neurons. Axonal injury further diminishes neuronal mTOR activity in these neurons. Forced upregulation of mTOR activity in corticospinal neurons by conditional deletion of Pten, a negative regulator of mTOR, enhances compensatory sprouting of uninjured CST axons and enables successful regeneration of a cohort of injured CST axons past a spinal cord lesion. Furthermore, these regenerating CST axons possess the ability to reform synapses in spinal segments distal to the injury. Thus, modulating neuronal intrinsic PTEN/mTOR activity represents a potential therapeutic strategy for promoting axon regeneration and functional repair after adult spinal cord injury (Liu, 2010).
Plexins are receptors for axonal guidance molecules semaphorins. The semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). This study examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. It is also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 alpha activity. Thus, it is proposed that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse (Oinuma, 2010).
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