myospheroid
Transcriptional regulation of integrins CD18, the beta chain of the leukocyte integrins, plays a crucial role in immune and inflammatory responses. CD18 is expressed exclusively by leukocytes, and it is transcriptionally regulated during the differentiation of myeloid cells. The ets factors (see Drosophila Pointed), PU.1 and GABP, bind to three ets sites in the CD18 promoter, which are essential for high level myeloid expression of CD18. Two binding sites have been identified for the transcription factor, Sp1 (see Drosophila Buttonhead); these binding sites flank the ets sites. Sp1 is
the only factor from myeloid cells that binds to these sites in a sequence-specific manner. Mutagenesis of these sites abrogates Sp1 binding and significantly reduces the activity of the transfected CD18 promoter in myeloid cells. Transfection of Sp1 into Drosophila Schneider cells, which otherwise lack Sp1, dramatically activates the CD18 promoter. GABP also activates the CD18 promoter in Schneider cells. Co-transfection of Sp1 and GABP activates CD18 more than
the sum of their individual effects, indicating that these factors cooperate to transcriptionally activate myeloid expression of CD18. These studies support a model of high level, lineage-restricted gene expression mediated by cooperative interactions between widely expressed transcription factors (Rosmarin, 1998).
Integrins and development Accumulating evidence indicates that the endometrial extracellular matrix (ECM) modulates trophoblast adhesion during mouse blastocyst implantation. In studies of adhesion-competent mouse blastocysts, it has been demonstrated that integrin-mediated fibronectin (FN)-binding activity on the apical surface of trophoblast cells is initially low, but becomes strengthened after embryos are exposed to FN. This study analyzes whether the ligand-induced upregulation of trophoblast adhesion to FN is mediated by integrin signaling. Direct evidence is provided that an intracellular signaling cascade is activated upon binding of FN to ß1- and ß3-class integrins, suggesting that the function of growth factors in regulating trophoblast invasion and early placenta formation may be augmented by uterine ECM components. The strengthening of adhesion to FN required integrin ligation, which rapidly elevates cytoplasmic-free Ca22+. Chelation of intracellular Ca2+ using BAPTA-AM, or inhibition of the Ca2+-dependent proteins, protein kinase C or calmodulin, significantly attenuates the effect of FN on binding activity. Furthermore, direct elevation of cytoplasmic Ca2+ levels with ionomycin upregulated FN-binding activity, demonstrating that Ca 2+ signaling is required and sufficient for strong adhesion to FN. Ca2+ signaling may induce protein trafficking, a known requirement for ligand-induced upregulation of FN-binding activity. Indeed, intracellular vesicles accumulated in adhesion-competent blastocysts, but were absent after exposure to either FN or ionomycin. These findings suggest that, during implantation, contact between peri-implantation blastocysts and FN elevates intracellular Ca2+, which strengthens trophoblast adhesion to ECM through protein redistribution (Wang, 2002).
Three novel beta integrin subunits are expressed during early development of the sea urchin
Strongylocentrotus purpuratus. The full cDNA sequence for one of these, betaG, bears a 59%
similarity to Drosophila betaPS and a 58% similarity to vertebrate integrins. betaG closely resembles
the beta1 subunit of vertebrates, particularly in the cytoplasmic domain where amino acids of the
human beta1 subunit implicated in cell adhesion and signaling are conserved. The betaG subunit is
detectable as a maternal, 7.5-kb transcript in eggs; expression peaks during gastrulation. betaG associates with at least two alpha
subunits in gastrula stage embryos. All
cells of the embryo express betaG prior to gastrulation. In gastrulae, hybridization of the probe is
highest in primary mesenchyme, secondary mesenchyme, the developing gut, and pigment cells. All cells of the blastulae express the protein at low levels; primary mesenchyme
cells express betaG after they enter the blastocoel. Expression of the protein appears to be
downregulated in the archenteron throughout gastrulation. betaG protein expression is also evident on
secondary mesenchyme as they ingress and migrate in the blastocoel. It is concluded that sea urchin
embryos express integrins that are structurally similar to those characterized in other animals. Because
betaG is expressed by migrating mesenchyme and yet is downregulated by rearranging epithelia, it is
suggested that this subunit has several functions during early development (Marsden, 1997).
Mesendoderm morphogenesis during gastrulation is described in the frog Xenopus laevis; the mechanics of these movements was investigated with tissue explants. When a dorsal marginal zone explant is plated onto fibronectin, the mesendoderm moves away from the dorsal axial tissues as an intact sheet. Mesendodermal cells within these explants display monopolar protrusive activity and radially intercalate during explant extension. Live time-lapse confocal sequences of actin dynamics at the margin of these extending explants prompt a proposal -- that integrin-mediated traction drives these movements. Integrin alpha5 ß1 recognition of the synergy site located within the type III9 repeat of fibronectin is required for mesendoderm extension. Normal mesendoderm morphogenesis occurs with a unique 'cup-shaped' geometry of the extending mesendodermal mantle and coincides with a higher rate of tissue extension than that seen in the simpler dorsal marginal zone explant. These higher rates can be reconstituted with 'in-the-round' configurations of several explants. Several mechanically based hypotheses are proposed to explain both the initial fibronectin-dependent extension of the mesendoderm and additional requirement of tissue geometry during the high-velocity closure of the mesendodermal mantle (Davidson, 2002).
Integrins regulate epithelial-mesenchymal interactions during
organogenesis. Mice with a mutation in the alpha8 gene do not express the integrin alpha8 beta1 and
exhibit profound deficits in kidney morphogenesis. In wild-type animals, inductive interactions between
the ureteric epithelium and metanephric mesenchyme are essential for kidney morphogenesis. In normal kidney development, the ureteric bud eminates from the Wolffian duct and invades the metanephric mesenchyme. These mesenchymal
cells are recruited into epithelial structures and are transformed into an epithelium consisting of distal tubules and proximal tubules that will mature into the secretory nephron. In
alpha8 mutant homozygotes, growth and branching of the ureteric bud and recruitment of mesenchymal
cells into epithelial structures are defective. Consistent with these phenotypes, alpha8 expression is
induced in mesenchymal cells upon contact with the ureteric epithelium. Since none of its previously identified
ligands appears likely to mediate the essential functions of alpha8 beta1 in kidney morphogenesis, an alpha8 beta1-alkaline phosphatase chimera was used to localize novel ligand(s) in the growing
ureter (Muller, 1997)
The sperm antigen fertilin alpha/beta and the integrin complex alpha6beta1 present on the oolemma are two of the most
promising candidates to mediate gamete interaction. During growth, the plasma membrane of both hamster and mouse
zona-free oocytes acquires the capacity to fuse with acrosome-reacted sperm when oocytes reach 25-30 mum
in diameter, suggesting changes in the membrane molecular composition. The present study has two aims: to determine (1)
the timing of gene expression of alpha6 and beta1 integrins and (2) the localization of these integrin subunits on the
plasma membrane in primordial germ cells and in oocytes during oogenesis. Both alpha6 and beta1 genes
are expressed in female germ cells during all the stages of development analyzed, from 10.5 to 18.5 d.p.c., during
oocyte growth, and in ovulated eggs. The alternatively spliced isoform alpha6B is expressed from 10.5 d.p.c., whereas
alpha6A begins to be expressed at 12.5 d.p.c., suggesting a different role for the two variants. In situ immunodetection
of alpha6 or beta1 shows a ring of fluorescence on the female germ cell plasma membrane for both integrins at 10.5
d.p.c., then the fluorescent signal becomes undetectable at 12.5 d.p.c. to reappear again, this time with a patchy
distribution, at 18.5 d.p.c. This pattern of localization is maintained in oocytes isolated from newborn individuals;
only when oocytes during growth reach about 25-30 mum in diameter does the fluorescence become
homogenous all around the whole oocyte surface. These data, although not conclusive, support the hypothesis of an
involvement of alpha6 and beta1 integrins in sperm-egg fusion (Zuccotti, 1998).
The earliest biochemical change detected during synaptogenesis is a local elimination of muscle basal
lamina proteins (heparan sulfate proteoglycans and laminin). To explore whether this provides any signal(s) that regulates postsynaptic differentiation, the effects of innervation were examined on the distribution of beta1-integrins, which are initially present in
scattered aggregates complexed with basal lamina ligands. These beta1-integrin aggregates disappear
along paths of nerve contact as their basal lamina ligands are eliminated. New accumulations of these
proteins then form during assembly of the postsynaptic apparatus. The new aggregates of beta1-integrin
at developing synapses form partly via a redistribution of mobile molecules on muscle surface. It is clear that the enzymatic degradation of muscle basal lamina proteins is closely coupled, spatially and temporally, with the disappearance of their associated beta1-integrin clusters: this strongly suggests that basal lamina degradation affects the cytoplasmic pathways that control the distribution of muscle beta1-integrins. This web of molecular interactions forms orderly signaling pathways that modulate (and are modulated by) interactions between integrin molecules and several cytoskeletal-associated proteins (such as talin, vinculin, alpha-actinin and paxillin), as well as several cytoplasmic enzymes, such as protein kinase C, p125FAK and pp60C-SRC (Anderson, 1997).
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of
central importance for the angiogenesis associated with cancers and other pathologies. Because
angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich
extracellular matrix, the possibility that VEGF promotes neovascularization through
regulation of collagen receptor expression was investigated. VEGF induces a 5- to 7-fold increase in dermal microvascular
EC surface protein expression of two collagen receptors (the alpha1beta1 and alpha2beta1
integrins), through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF does
not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen
binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibit attachment
of microvascular EC to collagen I, and alpha1-blocking Ab also inhibits attachment to collagen IV and
laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promotes cell spreading on
collagen I gels that is abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In
vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibits VEGF-driven
angiogenesis: the average cross-sectional area of individual new blood vessels is reduced 90% and average
total new vascular area is reduced 82% without detectable effects on the pre-existing vasculature.
These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important
mechanism by which VEGF promotes angiogenesis. It is also an indication that alpha1beta1 and alpha2beta1 antagonists may
prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies (Senger, 1997).
Alpha3beta1 integrin-deficient neonatal mice develop micro-blisters at the epidermal-dermal junction.
These micro-blisters are associated with poor basement membrane organization. The
effect of alpha3beta1-deficiency has been examined on other keratinocyte integrins, actin-associated proteins and F-actin organization. The absence of alpha3beta1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of
the basal keratinocytes of alpha3-null epidermis. Moreover, a higher concentration of actin-associated proteins such as
vinculin, talin, and alpha-actinin is seen at focal contact sites in the alpha3-deficient keratinocytes. These changes in focal contact
composition are not due to a change in steady-state levels of these proteins, but rather to reorganization due to alpha3beta1
deficiency. Apart from the loss of alpha3beta1 there is no change in expression of the other integrins expressed by the alpha3-null
keratinocytes. However, in functional assays, alpha3beta1 deficiency allows an increase in fibronectin and collagen type IV
receptor activities. Thus, these findings provide evidence of a role for alpha3beta1 in regulating stress fiber formation and as a
trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications
for the regulation of keratinocyte adhesion and migration during wound healing (Hodivala-Dilke, 1998).
Eyelid fusion normally occurs between E15.5 and E16.5 of mouse embryonic development and results from the migration of a
population of periderm-derived epithelial cells over the corneal surface. Cell migration is known to depend on extracellular
matrix receptors of the integrin family and to be regulated by growth factors. A failure of
eyelid fusion has been reported in mice that are homozygous null for the transforming growth factor alpha (TGF-alpha)
gene and in mice (invalpha5beta1) in which a transgenic alpha5beta1 integrin under the control of the involucrin
promoter is misexpressed in differentiating keratinocytes. Expression of the alpha2beta1,
alpha3beta1, alpha5beta1 and alpha6beta4 integrins during eyelid fusion was examined in wild-type embryos.
Selective upregulation of the alpha5beta1 integrin and its ligand, fibronectin, in the migrating eyelid tip cells was found. In
TGF-alpha null embryos, the failure of eyelid fusion is correlated with a failure to upregulate the alpha5beta1
integrin and fibronectin in the tip cells. Using beta-galactosidase as a reporter gene in transgenic mice, specific
activity of the involucrin promoter is observed in the eyelid tip cells. In invalpha5beta1 mice the transgenic human integrin is
overexpressed not only in the tip cells but throughout the eyelid epidermis. In contrast, the endogenous murine
alpha5beta1 integrin is only weakly expressed in the tip cells. It is speculated that selective and coordinated expression
of the alpha5beta1 integrin and fibronectin in eyelid tip cells is required for eyelid fusion and may be under the control
of growth factors that include TGF-alpha (Carroll, 1998).
Although spermatogenesis is essential for reproduction, little is known about spermatogonial stem cells. These cells provide the basis for spermatogenesis
throughout adult life by undergoing self-renewal and by providing progeny that differentiate into spermatozoa. A major impediment to an understanding of
the biology of these stem cells is the inability to distinguish them from spermatogonia that are committed to differentiation. The known
association of stem cells with basement membranes and a spermatogonial transplantation assay system were used to identify specific molecular markers on the stem
cell surface. Selection of mouse testis cells with anti-beta1- or anti-alpha6-integrin antibody, but not anti-c-kit antibody, produces cell populations with a
significantly enhanced ability to colonize recipient testes and generate donor cell-derived spermatogenesis. Spermatogonial stem
cell-associated antigens have been demonstrated by using an assay system based on biological function. Furthermore, the presence of surface integrins on spermatogonial stem cells
suggests that these cells share elements of a common molecular machinery with stem cells in other tissues (Shinohara, 1999).
Human epidermis contains two types of proliferative keratinocyte: the stem cell, which has a high self-renewal capacity and low
probability of terminal differentiation, and the transit-amplifying cell, the daughter of a stem cell that is destined to differentiate
within about three to five rounds of division. Keratinocytes with characteristics of stem cells persist in culture and
can be distinguished from transit-amplifying cells on the basis of the type of colony they form (2, 3). Colonies founded by stem
cells are able to self-renew, whereas transit-amplifying colonies contain fewer than 30-40 cells, all of which undergo terminal
differentiation. Human epidermal stem cells express higher levels of beta1 integrins
and are more adhesive than the keratinocytes that are destined to
differentiate. To investigate whether high beta1 integrin expression and
adhesiveness are essential for maintaining keratinocytes in the stem
cell compartment, a dominant-negative beta1 integrin
mutant, CD8beta1, was introduced into cultured human keratinocytes, thereby interfering
with beta1 integrin function. Surface beta1 integrin levels, adhesiveness, and mitogen-activated protein (MAP) kinase activation on
fibronectin were reduced, and exit from the stem cell compartment was
stimulated. Adhesiveness and proliferative potential are restored by
overexpressing wild-type beta1 integrin or by constitutive MAP kinase
activation. Conversely, a dominant-negative MAP kinase kinase 1 mutant
decreases adhesiveness and stem cell number in the absence of CD8beta1.
MAP kinase activation by alpha6beta4-mediated adhesion and mitogens is
normal in CD8beta1 cells, and constitutive MAP kinase activation does not
affect adhesion and proliferation of control keratinocytes. It is concluded
that beta1 integrins and MAP kinase cooperate to maintain the epidermal
stem cell compartment in vitro (Zhu, 1999).
The role of integrin-extracellular matrix interactions in the morphogenesis of ductal structures in vivo has been examined using the developing mouse mammary gland
as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and
become mitotically inactive. End buds are surrounded by a basement membrane, which contains laminin-1 and collagen IV. To address the role of
cell-matrix interactions in gland development, pellets containing function-perturbing anti-beta1 integrin, anti-alpha6 integrin, and anti-laminin antibodies, respectively,
were implanted into mammary glands at puberty. Blocking beta1 integrins dramatically reduces both the number of end buds per gland and the extent of the
mammary ductal network, compared with controls. These effects are specific to the end buds since the rest of the gland architecture remains intact. Reduced
development is still apparent after 6 days, but end buds subsequently reappear, indicating that the inhibition of beta1 integrins is reversible. Similar results
were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-alpha6 integrin antibody, suggesting that alpha6 is not
the important partner for beta1 in this system. The studies with beta1 integrin were confirmed in a culture model of ductal morphogenesis, where hepatocyte growth factor (HGF)-induced tubulogenesis is dependent on functional beta1 integrins. Thus integrins and HGF cooperate to regulate ductal
morphogenesis. It is proposed that both laminin and beta1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for
maintaining end bud structure and function in normal pubertal mammary gland development (Klinowska, 1999).
The dominant-negative function of chimeras between the beta1 cytoplasmic
domain and the extracellular domain of a nonintegrin protein could be
caused by competition with endogenous integrins for
intracellular factors. Alternatively, the chimeras
might deliver a signal that reduces the affinity of the
endogenous integrins for their ligands. The data favor a competition model in which beta1 signaling through MAPK is impaired. CD8beta1 expression results in reduced signaling to the MAPK cascade when keratinocytes are plated on fibronectin but such expression has no effect on MAPK activation in response to alpha6beta4-mediated adhesion or mitogens. Bypassing the beta1 integrin signaling defect by introduction of constitutively activated MAPKK1 into CD8beta1-expressing cells restores adhesiveness and increases the
proportion of stem cells. Conversely, a dominant-negative MAPKK1 mutant
is sufficient to reduce the adhesiveness and proliferative potential
of keratinocytes in the absence of CD8beta1. The ability of the
wild-type chicken beta1 construct to rescue proliferative potential and
MAPK activation in CD8beta1-expressing cells argues against an affinity
modulation signal from CD8beta1 and suggests that the strength of the
MAPK signal depends on the ratio of functional (i.e., wild-type) to
mutant (i.e., CD8beta1) receptors (Zhu, 1999).
There are at least three mechanisms by which beta1 integrin-mediated
adhesion can activate MAPK. In the first two, MAPK is activated
via Ras, either through FAK or Shc. In keratinocytes, FAK
phosphorylation is not inhibited by CD8beta1, as observed when similar
dominant-negative beta1 constructs are expressed in other cell types. The Shc-mediated pathway involves the interaction of the
transmembrane and juxtamembrane extracellular domains of integrin alpha subunits with caveolin, and it is difficult to envisage how
this process would be affected by overexpression of the beta1
cytoplasmic domain. The data presented in this paper favor a third mechanism that is
independent of Ras and FAK. It should also be noted that
in some contexts MAPK can down-regulate beta1 integrin function (Zhu, 1999).
The results show that a signaling pathway involving beta1 integrins and
MAPK controls epidermal stem cell fate in vitro and raise two important questions: why are high integrin levels required in order
for a keratinocyte to remain a stem cell, and how are those levels
controlled? Because ligand binding suppresses overt terminal
differentiation within the basal layer of the epidermis, high
surface levels of beta1 integrins could protect stem cells from
differentiation. Because the beta1 integrins have a pericellular distribution in stem and transit-amplifying cells, the
proportion of surface integrins in contact with the basement membrane
will be similar in both cell populations. It therefore seems likely
that it is the absolute number of occupied receptors that is important
for the protective effect (Zhu, 1999).
The extent to which stem cell behavior is preprogrammed or
environmentally regulated has long been a subject for debate. Although epidermal stem cell number is subject to autoregulation,
it is believed that environmental factors, specifically the composition of
the basement membrane, could also be key determinants. Stimulatory or
inhibitory input into the beta1 integrin/MAPK pathway at different
levels could provide a mechanism by which the environment influences
the proliferative capacity of basal keratinocytes. ECM proteins can
modulate beta1 integrin expression and activation, and local
variation in the composition of the basement membrane could thus
play a role in establishing and maintaining the patterned distribution
of stem cells within the epidermal basal layer (Zhu, 1999).
The major epidermal integrins are alpha3ß1 and hemidesmosome-specific alpha6ß4; both share laminin 5 as ligand. Keratinocyte culture studies implicate both integrins in adhesion, proliferation, and stem cell maintenance and suggest unique roles for alphaß1 integrins in migration and terminal differentiation. In mice, however, whereas ablation of alpha6 or ß4 results in loss of
hemidesmosomes, epidermal polarity, and basement membrane (BM) attachment, ablation of alpha3 only generates microblistering due to localized internal shearing of BM. Using conditional knockout technology to ablate ß1 in skin epithelium, biological roles for alphaß1 integrins have been uncovered that are not predicted from either the alpha3 knockout or from in vitro studies. In contrast to alpha3 null
mice, ß1 mutant mice exhibit severe skin blistering and hair defects, accompanied by massive failure of BM assembly/organization,
hemidesmosome instability, and a failure of hair follicle keratinocytes to remodel BM and invaginate into the dermis. Although
epidermal proliferation is impaired, a spatial and temporal program of terminal differentiation is executed. These results indicate that
ß1's minor partners in skin are important, and together, alphaß1 integrins are required not only for extracellular matrix assembly
but also for BM formation. This, in turn, is required for hemidesmosome stability, epidermal proliferation, and hair follicle
morphogenesis. However, ß1 downregulation does not provide the trigger to terminally differentiate (Raghavan, 2000).
In summary, the conditional ablation of the ß1 integrin gene in mouse epidermis has provided major new insights into the functions of alphaß1 integrins and into the differential roles of ß1 and ß4 integrins in the skin. The findings argue against an essential role for alphaß1 integrins in regulating the spatial and temporal program of epidermal terminal differentiation, a function predicted from keratinocyte culture studies conducted with mutant ß1 transgenes. In contrast, the results underscore a critical role for ß1 in maintaining proliferative potential in developing skin epithelium and provide compelling support for previous in vitro studies correlating proliferative potential with surface levels of ß1 integrins in keratinocytes. Perhaps most interesting in this regard is that these findings reveal that the underlying reason for this long-standing observation is likely the unique ability of alphaß1 integrins to assemble BM. This insight was not obtained from the alpha3 knockout, where BM was largely intact and proliferation unaffected, although some clues to potential roles for alphaß1 integrins in BM assembly have emerged from studies on other tissues. The dramatic defect in the BM, not seen yet for any other epidermal integrin knockout, enabled the uncovering of an unanticipated new role for alphaß1 integrins in hemidesmosome assembly/stabilization and in hair follicle invagination into the underlying dermis. The challenge for the future will be to dissect the molecular pathways used by alphaß1 integrins in orchestrating these events (Raghavan, 2000).
Myelination represents a remarkable example of cell specialization and cell-cell interaction in development.
During this process, axons are wrapped by concentric layers of cell membrane derived either from central
nervous system (CNS) oligodendrocytes or peripheral nervous system Schwann cells. In the CNS,
oligodendrocytes elaborate a membranous extension with an area of more than 1000 times that of the cell body. The mechanisms regulating this change in cell shape remain poorly understood. Signaling mechanisms
regulated by cell surface adhesion receptors of the integrin family represent likely candidates. Integrins link the
extracellular environment of the cell with both intracellular signaling molecules and the cytoskeleton and have
been shown to regulate the activity of GTPases implicated in the control of cell shape. Oligodendrocytes and their precursors express a limited repertoire of integrins. One of these, the alpha6ß1 laminin receptor, can interact with laminin-2 substrates to enhance oligodendrocyte myelin membrane formation in cell culture. However, these experiments do not address the important question of integrin function during myelination in vivo, nor do they define the respective roles of the subunits in the signaling pathways involved. A dominant-negative approach has been used to provide evidence that ß1 integrin function is
required for myelination in vivo and chimeric integrins have been used to dissect apart the roles of the extracellular and cytoplasmic domains of the alpha6 subunit in the signaling pathways of myelination (Relvas, 2001).
In order to assess the in vivo function of integrins containing the ß8 integrin subunit, ß8-deficient mice have been generated. Ablation of ß8 results in embryonic or perinatal lethality with profound defects in vascular development. Sixty-five percent of integrin ß8-deficient embryos die at midgestation, with evidence of insufficient vascularization of the placenta and yolk sac. The remaining 35% die shortly after birth with extensive intracerebral hemorrhage. Examination of brain tissue from integrin ß8-deficient embryos reveals abnormal vascular morphogenesis resulting in distended and leaky capillary vessels, as well as aberrant brain capillary patterning. In addition, endothelial cell hyperplasia is found in these mutant brains. Expression studies show that integrin ß8 transcripts are localized in endodermal cells surrounding endothelium in the yolk sac and in periventricular cells of the neuroepithelium in the brain. It is proposed that integrin ß8 is required for vascular morphogenesis by providing proper cues for capillary growth in both yolk sac and embryonic brain. This study thus identifies a molecule crucial for vascular patterning in embryonic yolk sac and brain (Zhu, 2002).
ß1 integrins are highly expressed on chondrocytes, where they mediate adhesion to cartilage matrix proteins. To assess the functions of ß1 integrin during skeletogenesis, the ß1 integrin gene was inactivated in chondrocytes. These mutant mice develop a chondrodysplasia of various severity. ß1-deficient chondrocytes have an abnormal shape and fail to arrange into columns in the growth plate. This is caused by a lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. The G1/S defect is, at least partially, caused by overexpression of Fgfr3, nuclear translocation of Stat1/Stat5a, and up-regulation of the cell cycle inhibitors p16 and p21. Altogether these findings establish that ß1-integrin-dependent motility and proliferation of chondrocytes are mandatory events for endochondral bone formation to occur (Aszodi, 2003).
A major challenge confronting developmental cell biologists is to understand how individual cell behaviors lead to global tissue organization. Taking advantage of an endothelial cell-specific marker and scanning time-lapse microscopy, the formation of the primary vascular pattern during avian vasculogenesis has been examined. Five types of distinguishable endothelial cell motion are observed during formation of a vascular plexus: (1) global tissue deformations that passively convect endothelial cells; (2) vascular drift, a sheet-like medial translocation of the entire vascular plexus; (3) structural rearrangements, such as vascular fusion; (4) individual cell migration along existing endothelial structures, and (5) cell process extension into avascular areas, resulting in new links within the plexus. The last four types of motion are quantified and found to be reduced in the presence of an alphavß3 integrin inhibitor. These dynamic cell motility data result in new hypotheses regarding primordial endothelial cell behavior during embryonic vasculogenesis (Rupp, 2004).
The control point by which chondrocytes take the decision between the
cartilage differentiation program or the joint formation program is unknown.
The effect of alpha5ß1 integrin inhibitors
and bone morphogenetic protein (BMP) on joint formation was investigated. Blocking of alpha5ß1 integrin by specific antibodies or RGD peptide
(arginine-glycine-aspartic acid) induces inhibition of pre-hypertrophic
chondrocyte differentiation and ectopic joint formation between proliferating
chondrocytes and hypertrophic chondrocytes. Ectopic joint expresses Wnt14,
Gdf5, chordin, autotaxin, type I collagen and CD44, while expression of
Indian hedgehog and type II collagen is downregulated in cartilage.
Expression of these interzone markers confirmed that the new structure is a
new joint being formed. In the presence of BMP7, inhibition of alpha5ß1
integrin function still induces the formation of the ectopic joint between
proliferating chondrocytes and hypertrophic chondrocytes. By contrast,
misexpression of alpha5ß1 integrin results in fusion of joints and
formation of pre-hypertrophic chondrocytes. These facts indicate that the
decision of which cell fate to make pre-joint or pre-hypertrophic is made on
the basis of the presence or absence of alpha5ß1 integrin on
chondrocytes (Garciadiego-Cázares, 2004).
Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling play essential roles in neural development. Echistatin, an RGD-containing short monomeric disintegrin, was used to investigate the role of integrin-mediated adhesion and signaling during retinal development in Xenopus. Application of echistatin to Xenopus retinal-derived XR1 glial cells inhibited the three stages of integrin-mediated adhesion: cell attachment, cell spreading, and formation of focal adhesions and stress fibers. XR1 cell attachment and spreading increases tyrosine phosphorylation of paxillin, a focal adhesion associated protein, while echistatin significantly decreases phosphorylation levels of paxillin. Application of echistatin or ß1 integrin function-blocking antibody to the embryonic Xenopus retina disrupts retinal lamination and produces rosette structures with ectopic photoreceptors in the outer retina. These results indicate that integrin-mediated cell-ECM interactions play a critical role in cell adhesion, migration, and morphogenesis during vertebrate retinal development (Lia, 2004).
Integrin receptors for the extracellular matrix and receptor tyrosine kinase growth factor receptors represent two of the major families of receptors that transduce into cells information about the surrounding environment. Wnt proteins are a major family of signaling molecules that regulate morphogenetic events. There is presently little understanding of how the expression of Wnt genes themselves is regulated. This study demonstrates that alpha3beta1 integrin, a major laminin receptor involved in the development of the kidney, and c-Met, the receptor for hepatocyte growth factor, signal coordinately to regulate the expression of Wnt7b in the mouse. Wnt signals in turn appear to regulate epithelial cell survival in the papilla of the developing kidney, allowing for the elongation of epithelial tubules to form a mature papilla. Together, these results demonstrate how signals from integrins and growth factor receptors can be integrated to regulate the expression of an important family of signaling molecules so as to regulate morphogenetic events (Liu, 2009).
Growth and expansion of ventricular chambers is essential during heart development and is achieved by proliferation of cardiac progenitors. Adult cardiomyocytes, by contrast, achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Using a coculture system, it was found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. Fibronectin, collagen, and heparin-binding EGF-like growth factor were identified as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. Myocardial β1-integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of β1-integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation. The recent report of proliferative defects upon cardiac deletion of focal adhesion kinase (Peng, 2008), an important mediator between β1 integrin and ERK1/2 or PI3K/Akt, is consistent with the findings. However, the heart size of Nkx2.5-Cre/Itgb1flox/flox mice, deficient in the laminin-specific receptor, was generally smaller than wild-type littermates, but that of FAK knockout mice were not. FGF signaling, also downstream of β1 integrin, is known to be critical for heart size (Ieda, 2009).
Vascular smooth muscle cells (VSMCs) form contractile layers around larger blood vessels in a process that is essential for the formation of a fully functional vasculature. This study shows that integrin-linked kinase (ILK) is required for the formation of a unitary layer of aligned VSMCs around arterioles and the regulation of blood vessel constriction in mice. In the absence of ILK, activated Rho/ROCK signaling induces the elevated phosphorylation of myosin light chain leading to abnormally enhanced VSMC contraction in vitro and in vivo. These findings identify ILK as a key component regulating vascular wall formation by negatively modulating VSMC contractility (Kogata, 2009).
It has been reported that the major β integrin subunit β1 contributes to vascular smooth muscle function (Abraham, 2008). Gene inactivation of β1 integrin in mice impairs VSMC spreading and differentiation, and increases VSMC proliferation leading postnatal lethality (Abraham, 2008). This study reports that ILK plays a fundamental role in developing vascular wall assembly that is distinct from that of β1 integrin. Mice lacking ILK expression in vascular wall cells fail to assemble their VSMCs into a unitary layer, which results in defective vascular remodeling and embryonic lethality. Moreover, this study shows that loss of ILK causes increased VSMC contractility due to elevated myosin light-chain phosphorylation through activation of Rho/ROCK signaling (Kogata, 2009).
β1 integrins are important regulators of vascular differentiation and development, as their endothelial-specific deletion results in embryonic lethality. This study investigated the molecular mechanisms underlying the prominent vascular abnormalities that occur in the absence of β1 integrins. Because of the early embryonic lethality of knockout mice, endothelial cell and vessel development were studied in β1-integrin-deficient murine embryonic stem cells to gain novel insights into the role of β1 integrins in vasculo-angiogenesis. Vessel development was found to be strongly defective in the mutant embryoid bodies (EBs), since only primitive and short sprouts developed from clusters of vascular precursors in β1 integrin-/- EBs, whereas complex network formation of endothelial tubes was observed in wild-type EBs. The vascular defect was due to deficient β1 integrin expression in endothelial cells, as its endothelial-specific re-expression rescued the phenotype entirely. The mechanism responsible for defective vessel formation was found to be reduced endothelial cell maturation, migration and elongation. Moreover, the lower number of endothelial cells in β1 integrin-/- EBs was due to an increased apoptosis versus proliferation rate. The enhanced apoptosis and proliferation of β1 integrin-/- endothelial cells was related to the elevation of peNOS and pAKT signaling molecules, respectively. These data demonstrate that endothelial β1 integrins are determinants of vessel formation and that this effect is mediated via different signaling pathways (Malan, 2010).
Maintenance of single-layered endothelium, squamous endothelial cell shape, and formation of a patent vascular lumen all require defined endothelial cell polarity. Loss of β1 integrin (Itgb1) in nascent endothelium leads to disruption of arterial endothelial cell polarity and lumen formation. The loss of polarity is manifested as cuboidal-shaped endothelial cells with dysregulated levels and mislocalization of normally polarized cell-cell adhesion molecules, as well as decreased expression of the polarity gene Par3 (pard3). β1 integrin and Par3 are both localized to the endothelial layer, with preferential expression of Par3 in arterial endothelium. Luminal occlusion is also exclusively noted in arteries, and is partially rescued by replacement of Par3 protein in β1-deficient vessels. Combined, these findings demonstrate that β1 integrin functions upstream of Par3 as part of a molecular cascade required for endothelial cell polarity and lumen formation (Zovein, 2010).
Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons to ensure fast propagation of action potentials. beta1 integrins regulate the myelination of peripheral nerves, but their function during the myelination of axonal tracts in the CNS is unclear. This study shows that genetically modified mice lacking beta1 integrins in the CNS present a deficit in myelination but no defects in the development of the oligodendroglial lineage. Instead, in vitro data show that beta1 integrins regulate the outgrowth of myelin sheaths. Oligodendrocytes derived from mutant mice are unable to efficiently extend myelin sheets and fail to activate AKT (also known as AKT1), a kinase that is crucial for axonal ensheathment. The inhibition of PTEN, a negative regulator of AKT, or the expression of a constitutively active form of AKT restores myelin outgrowth in cultured beta1-deficient oligodendrocytes. These data suggest that beta1 integrins play an instructive role in CNS myelination by promoting myelin wrapping in a process that depends on AKT (Barros, 2009).
Integrins and neuronal migration Migrating neural crest cells adhere to fibronectin in an integrin-dependent manner while maintaining
reduced N-cadherin-mediated intercellular contacts (see Drosophila Cadherin-N). In
these cells, the control of N-cadherin may rely directly on the activity of integrins involved in the
process of cell motion. Prevention of neural crest cell migration using RGD peptides or antibodies to
fibronectin and to beta1 and beta3 integrins causes rapid N-cadherin-mediated cell clustering.
Restoration of stable intercellular contacts results essentially from the recruitment of an intracellular
pool of N-cadherin molecules that accumulate into adherens junctions in tight association with the
cytoskeleton and not from the redistribution of a preexisting pool of surface N-cadherin molecules. Agents that cause elevation of intracellular Ca2+ after entry across the plasma membrane
are potent inhibitors of cell aggregation and reduced the N-cadherin- mediated junctions in the cells.
Elevated serine/ threonine phosphorylation of catenins associated with N-cadherin accompany
the restoration of intercellular contacts. These results indicate that in migrating neural crest cells, beta1
and beta3 integrins are at the origin of a cascade of signaling events that involves transmembrane Ca2+
fluxes, followed by activation of phosphatases and kinases that ultimately control the surface
distribution and activity of N-cadherin. Cell aggreation is correlated with changes in beta-catenin phosphorylation. Such a direct coupling between adhesion receptors by means of
intracellular signals may be significant for the coordinated interplay between cell-cell and
cell-substratum adhesion that occurs during embryonic development, in wound healing, and during
tumor invasion and metastasis (Monier-Gavelle, 1997).
During early embryonic development, cranial neural crest cells emerge from the developing mid- and hind-brain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. Fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and 'synergy' sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Using a function blocking monoclonal antibody, it has been shown that cranial neural crest cell migration on fibronectin requires the integrin alpha5ß1 (Alfandari, 2003).
Proliferation and tangential migration of neural precursor cells are essential determinants of CNS
development. Migration of neural precursors and committed progeny occurs in two major axes relative to the neuroepthelium; radial and tangential. Tangential migration is seen within the neuroepithelium and in the rostral migration stream leading to the olfactory bulb, and has been described both for neuronal precursors and for less differentiated neuroepithelial precursor cells. Examples of radial and tangential migrations show that they differ in their substrata for movement. Radial migration occurs along glial fibers that act as a scaffold to guide migrating cells. In contrast, cells undergoing tangential migration appear to not always use a different cell type as a scaffold for migration. Neuronal precursors within the rostral migratory stream migrate in contact with one another without a glial scaffold, a form of migration that has been termed chain migration. Cell culture models have been established for both proliferation and tangential migration processes using neural precursor cells
grown as neurospheres. The pattern of migration observe in these cells is homotypic and occurs in
the absence of a glial or neuronal scaffold, and is therefore equivalent to that previously described as chain
migration. To determine the role of integrins in proliferation and migration, the
expression pattern of integrins on neurosphere cells has been analyzed and subsequent blocking peptide and antibody
experiments have been performed. Neurosphere cells express five major integrins, alpha5beta1, alpha6Abeta1, alphavbeta1, alphavbeta5 and alphavbeta8, in addition to expressing low levels of alpha6Bbeta1. Chain
migration is inhibited by blocking the alpha6beta1 integrin. In contrast, proliferation is inhibited by
blocking the other beta1 integrins, alphavbeta1 and alpha5beta1. These results show that integrins are
important regulators of neural precursor cell behaviour, with distinct beta1 integrins regulating proliferation
and migration. They also demonstrate a novel role for the alpha6beta1 integrin in the cell-cell interactions
underlying homotypic chain migration (Jacques, 1998).
Integrins are transmembrane receptors that are known to interact with the extracellular matrix and to be required for migration, proliferation, differentiation and apoptosis. Mice have been generated with a neural crest cell-specific deletion of the ß1-integrin gene to analyse the role of ß1-integrins in neural crest cell migration and differentiation. This targeted mutation caused death within a month of birth. The loss of ß1-integrins from the embryo delayed the migration of Schwann cells along axons and induced multiple defects in spinal nerve arborisation and morphology. There was an almost complete absence of Schwann cells and sensory axon segregation and defective maturation in neuromuscular synaptogenesis. Thus, ß1-integrins are important for the control of embryonic and postnatal peripheral nervous system development (Pietri, 2004).
Alpha3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in alpha3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of alpha3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons of alpha3 integrin mutant mice expressing EGFP were misplaced. These results suggest that alpha3beta1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex (Schmid, 2005).
Integrins and axon guidance Radial sorting allows the segregation of axons by a single Schwann cell (SC) and is a prerequisite for myelination during peripheral nerve development. Radial sorting is impaired in models of human diseases, congenital muscular dystrophy (MDC) 1A, MDC1D and Fukuyama, owing to loss-of-function mutations in the genes coding for laminin α2, Large or fukutin glycosyltransferases, respectively. It is not clear which receptor(s) are activated by laminin 211, or glycosylated by Large and fukutin during sorting. Candidates are αβ1 integrins, because their absence phenocopies laminin and glycosyltransferase deficiency, but the topography of the phenotypes is different and β1 integrins are not substrates for Large and fukutin. By contrast, deletion of the Large and fukutin substrate dystroglycan does not result in radial sorting defects. This study shows that absence of dystroglycan in a specific genetic background causes sorting defects with topography identical to that of laminin 211 mutants, and recapitulating the MDC1A, MDC1D and Fukuyama phenotypes. By epistasis studies in mice lacking one or both receptors in SCs, it was showm that only absence of β1 integrins impairs proliferation and survival, and arrests radial sorting at early stages, that β1 integrins and dystroglycan activate different pathways, and that the absence of both molecules is synergistic. Thus, the function of dystroglycan and β1 integrins is not redundant, but is sequential. These data identify dystroglycan as a functional laminin 211 receptor during axonal sorting and the key substrate relevant to the pathogenesis of glycosyltransferase congenital muscular dystrophies (Berti, 2011).
Integrins and synaptic plasticity In order to stabilize changes in synaptic strength, neurons activate a program of gene
expression that results in alterations of their molecular composition and structure. Fnk and Snk, two members of the polo family of cell cycle associated
kinases, are co-opted by the brain to serve in this program.
All members of
this family are characterized by the same domain topology; alignment in a phylogenetic tree indicates that the
polo-like kinases diverged before the subfamily of calmodulin kinase-related genes developed. Comparison of the deduced amino acid sequences of human Prk, rat Fnk and mouse Fnk shows that the three polypeptides are ~90% identical except for a 17-amino-acid insertion present in rat
Fnk and human Prk. Sequences encoding the 17-amino-acid insertion of human and rat Fnk are also present in
mouse mRNA from NIH-3T3 fibroblasts and are flanked by two
alternative 5' consensus splice sites in the mouse genomic sequence. This suggests that the published sequence of mouse
Fnk most likely represents a splice variant. Rat Fnk shares ~50% sequence identity with rat Snk which is ~90%
identical to the mouse homolog. The N-terminal half of Fnk and Snk harbors a
serine/threonine-specific kinase domain including all 11 subdomains described as specific for serine/threonine kinases. The C-terminal half contains a 30-amino-acid domain referred to as the polo-box,
which is highly conserved in all family members. This motif has not been described in any other protein and its
function has not been determined (Kauselmann, 1999).
Stimuli that produce synaptic
plasticity, including those that evoke long-term potentiation (LTP), dramatically increase
levels of both Fnk and Snk mRNAs. Induced Fnk and Snk proteins are targeted to the dendrites of activated neurons,
suggesting that they mediate phosphorylation of proteins in this compartment. Moreover, a conserved C-terminal
domain in these kinases is shown to interact specifically with Cib, a Ca2+ and integrin-binding protein. Cib shares greater than 27% amino acid identity with calcineurin B, the regulatory subunit of calcineurin (phosphatase 2B), and has
a similar identity to calmodulin. Like calcineurin B and calmodulin, Cib contains EF-hand motifs
responsible for Ca2+ binding. The structural similarity of Cib to calmodulin and to calcineurin B raises the possibility that
Cib might be a regulatory subunit of Snk or Fnk or, alternatively, the regulatory subunit of a phosphatase whose catalytic
subunit has not yet been identified. In this case, the association of a kinase-phosphatase complex could facilitate rapid
cycles of phosphorylation or result in an autocatalytic loop of kinase-phosphatase activation. Together, these
studies suggest a novel signal transduction mechanism in the stabilization of long-term synaptic plasticity (Kauselmann, 1999).
Active zones are the sites along nerve terminals where synaptic vesicles dock and undergo calcium-dependent exocytosis during synaptic transmission. Alpha3beta1 integrin is concentrated at the active zones of Xenopus motor nerve terminals. Because integrins can link extracellular
matrix molecules to cytoskeletal elements and participate in the formation of signaling complexes, the localization of integrin at active zones suggests that it may play a role in the adhesion of the nerve terminals to the synaptic basal lamina, in the formation and maintenance of active zones, and in some of the events associated with calcium-dependent exocytosis of neurotransmitter. These findings also indicate that the integrin composition of the terminal Schwann cells differs from that of the motor nerve terminals, and this may account at least in part for differences in their adhesiveness to the synaptic basal lamina (Cohen, 2000).
Integrins, cell survival, cellular senescence and apoptosis The role of integrins in leukocyte apoptosis is unclear: some studies suggest enhancement, others inhibition. ß2-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of alphaMß2 delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFalpha, or UV irradiation, ligation of active alphaMß2 results in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins does not show this proapoptotic effect and continues to inhibit apoptosis. This discrepancy can be attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive alphaMß2 is capable of stimulating the kinases ERK and Akt. Activated alphaMß2 stimulates Akt, but not ERK. When proapoptotic stimuli are combined with either alphaMß2 clustering or activation, Akt activity is blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive alphaMß2 continues to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, ß2-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes (Whitlock, 2000).
Proliferation and survival of Schwann cells are important for nerve development and for disease processes in peripheral
nerves. Embryos lacking alpha4- or alpha5-integrins have been examined and it is shown here that these integrins contribute to the control of glial cell numbers. To overcome early embryonic lethality an explant and grafting system that allows the study of isolated
glial progenitor cells both in vitro and in vivo was used. Schwann cells differentiate in the absence of alpha5 but their numbers
and the proliferation rate of early progenitor cells are reduced, suggesting that alpha5 is essential for normal proliferation.
Survival, rather than proliferation, is compromised in alpha4-deficient explants. Conditional immortalization allowed further
characterization and revealed that alpha4 contributes to survival in a cell-density-dependent fashion. In addition, transplants
into chicken embryos used to analyze in vivo cell migration show that cell death occurs mainly in highly motile, individually migrating cells. The cell death patterns in vitro and in vivo argue that alpha4-integrins play a role in survival during cell migration. Neural crest migration has been suggested to require these integrins; however, no defects in migration were
observed in the absence of alpha4 or alpha5. It is concluded that integrins can complement growth factors in the control of glial cell
numbers (Haack, 2001).
The reduced proliferation rate in clonal culture and the
reduced number of mature Schwann cells in culture show
that alpha5 is required for efficient proliferation of glial progenitor
cells. In addition, the inability of large T antigen to rescue alpha5-null
cells is a strong indication that cell-matrix interactions are
crucial. The expression profile of FN and alpha5 correlates well
with phases of proliferation in developing nerves and during
nerve regeneration, in which both FN and alpha5 are upregulated
at the site of injury. FN is strongly mitogenic when added to the
culture medium and is specifically upregulated during the transition from slowly
to rapidly dividing cells. Thus, FN appears to be important for glial proliferation, likely to be mediated by integrins. Based on the strong proliferative
defect of alpha5-deficient cells, it is suggest that alpha5beta1 is the
predominant receptor transmitting FN-mediated growth stimuli in Schwann cells. alpha5beta1 has been associated with proliferation in a number of other cell types although the signaling pathways remain incompletely understood (Haack, 2001).
PTEN is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. Somatic mutations of PTEN are found in a number of human malignancies, and loss of expression, or mutational inactivation of PTEN, leads to the constitutive activation of protein kinase B (PKB)/Akt via enhanced phosphorylation of Thr-308 and Ser-473. The integrin-linked kinase (ILK) can phosphorylate PKB/Akt on Ser-473 in a phosphoinositide phospholipid-dependent manner. The activity of ILK is constitutively elevated in a serum- and anchorage-independent manner in PTEN-mutant cells, and transfection of wild-type (WT) PTEN into these cells inhibits ILK activity. Transfection of a kinase-deficient, dominant-negative form of ILK or exposure to a small molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473, but not on Thr-308, in the PTEN-mutant prostate carcinoma cell lines PC-3 and LNCaP. Transfection of dominant-negative ILK and WT PTEN into these cells also results in the inhibition of PKB/Akt kinase activity. Furthermore, dominant-negative ILK or WT PTEN induces G(1) phase cycle arrest and enhanced apoptosis. Together, these data demonstrate a critical role for ILK in PTEN-dependent cell cycle regulation and survival and indicate that inhibition of ILK may be of significant value in PTEN-mutant tumor therapy (Persad, 2000).
In the retina, integrins in the ß1 family have been shown to be important in many phases of neuronal development, particularly neuroblast migration and axon outgrowth. However, the functions of specific integrin heterodimers are not well defined. In this study, the functions of ß1 integrins in developing chicken retina were investigated by expression of a dominant-negative ß1A construct using a replication-competent retrovirus. Inhibition of integrins using this approach resulted in alteration of cell morphology and increased apoptosis, but did not preclude migration and axon elongation. In an attempt to identify which specific ß1 heterodimer was important, expression and function of the a4ß1 heterodimer were also investigated. At early developmental stages, a4 protein and mRNA were detected in undifferentiated neuroblasts throughout the retina. At later stages, expression is confined to retinal ganglion cells (RGCs) and amacrine cells. A small molecule antagonist of a4 integrins inhibits neurite outgrowth on recombinant soluble vascular cell adhesion molecule-1 (VCAM-1), a known ligand of a4ß1. Introduction of a4 antagonist in vivo gives rise to increased apoptosis and leads to a thinning of the retina and reduced numbers of retinal ganglion cells (RGCs). It is concluded that the integrin a4ß1 is important for survival of developing retinal neurons, including RGCs (Leu, 2004).
Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. This study found that ITGB3 (integrin β 3 or β3; see Drosophila Myospheroid) is regulated by the Polycomb protein CBX7. β3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor β (TGF-β) pathway in a cell-autonomous and non-cell-autonomous manner. β3 levels are dynamically increased during oncogene-induced senescence (OIS) through CBX7 Polycomb regulation, and downregulation of β3 levels overrides OIS and therapy-induced senescence (TIS), independently of its ligand-binding activity. Moreover, cilengitide, an αvβ3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP) without affecting proliferation. Finally, this study shows an increase in β3 levels in a subset of tissues during aging. Altogether, these data show that integrin β3 subunit is a marker and regulator of senescence (Rapisarda, 2017).
Integrins are ubiquitous transmembrane receptors that play crucial roles in cell-cell and cell-matrix interactions. In this study, the effects of the loss of ß1 integrins in keratinocytes in vitro and during cutaneous wound repair has been determined. Flow cytometry of cultured ß1-deficient keratinocytes has confirmed the absence of ß1 integrins and shows downregulation of alpha6ß4 but not of alphav integrins. ß1-null keratinocytes are characterized by poor adhesion to various substrates, by a reduced proliferation rate and by a strongly impaired migratory capacity. In vivo, the loss of ß1 integrins in keratinocytes causes a severe defect in wound healing. ß1-null keratinocytes show impaired migration and are more densely packed in the hyperproliferative epithelium. Surprisingly, their proliferation rate is not reduced in early wounds and even increases in late wounds. The failure in re-epithelialization results in a prolonged inflammatory response, leading to dramatic alterations in the expression of important wound-regulated genes. Ultimately, ß1-deficient epidermis does cover the wound bed, but the epithelial architecture is abnormal. These findings demonstrate a crucial role of ß1 integrins in keratinocyte migration and wound re-epithelialisation (Grose, 2002).
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