Epidermal Growth Factor Module Research Articles

Human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium.

To investigate human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium (CM). A human corneal epithelial cell line (HCE-T), human corneal fibroblasts (HCF) and keratoconus fibroblasts (KC-HCF) have been used. Twenty-four hours after RB-PDT (0.001% RB concentration, 565 nm wavelength illumination, 0.17 J/cm2 fluence) cell migration rate using scratch assay and growth factor concentrations in the cell culture supernatant using ELISA have been determined. In addition, the effect of CM has been observed. RB-PDT significantly reduced migration rate in all cell types, compared to controls (p≤0.02). Migration rate of HCE-T cultures without RB-PDT (untreated) was significantly higher using HCF CM after RB-PDT, than using HCF CM without RB-PDT (p<0.01). Similarly, untreated HCF displayed a significantly increased migration rate with HCE-T CM after RB-PDT, compared to HCE-T CM without treatment (p<0.01). Furthermore, illumination alone and RB-PDT significantly decreased keratinocyte growth factor (KGF) concentration in HCF and KC-HCF supernatant, and RB-PDT significantly decreased soluble N-Cadherin (SN-Cad) concentration in HCF supernatant, compared to controls (p<0.01 for all). In HCE-T CM, RB-PDT increased hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGFb) concentration (p≤0.02), while decreasing transforming growth factor β (TGF-β) concentration (p<0.01). FGFb concentration increased (p<0.0001) and TGF-β concentration decreased (p<0.0001) in HCF CM, by RB-PDT. Epidermal growth factor (EGF), HGF, and TGF-β concentration decreased (p≤0.03) and FGFb concentration increased (p<0.01) in KC-HCF CM, using RB-PDT. HCE-T, HCF and KC-HCF migration rate is reduced 24 hours after RB-PDT. In contrast, HCE-T migration is enhanced using HCF CM after RB-PDT, and HCF migration rate is increased through HCE-T CM following RB-PDT. Modulation of EGF, KGF, HGF, FGFb, TGF-β and N-Cadherin secretion through RB-PDT may play an important role in corneal wound healing.

Repurposed anti-cancer epidermal growth factor receptor inhibitors: mechanisms of neuroprotective effects in Alzheimer's disease.

Numerous molecular mechanisms are being examined in an attempt to discover disease-modifying drugs to slow down the underlying neurodegeneration in Alzheimer’s disease. Recent studies have shown the beneficial effects of epidermal growth factor receptor inhibitors on the enhancement of behavioral and pathological sequelae in Alzheimer’s disease. Despite the promising effects of epidermal growth factor receptor inhibitors in Alzheimer’s disease, there is no irrefutable neuroprotective evidence in well-established animal models using epidermal growth factor receptor inhibitors due to many un-explored downstream signaling pathways. This caused controversy about the potential involvement of epidermal growth factor receptor inhibitors in any prospective clinical trial. In this review, the mystery beyond the under-investigation of epidermal growth factor receptor in Alzheimer’s disease will be discussed. Furthermore, their molecular mechanisms in neurodegeneration will be explained. Also, we will shed light on SARS-COVID-19 induced neurological manifestations mediated by epidermal growth factor modulation. Finally, we will discuss future perspectives and under-examined epidermal growth factor receptor downstream signaling pathways that warrant more exploration. We conclude that epidermal growth factor receptor inhibitors are novel effective therapeutic approaches that require further research in attempts to be repositioned in the delay of Alzheimer’s disease progression.

Structural studies of reelin N-terminal region provides insights into a unique structural arrangement and functional multimerization.

The large, secreted glycoprotein reelin regulates embryonic brain development as well as adult brain functions. Although reelin binds to its receptors via its central part, the N-terminal region directs multimer formation and is critical for efficient signal transduction. In fact, the inhibitory antibody CR-50 interacts with the N-terminal region and prevents higher-order multimerization and signalling. Reelin is a multidomain protein in which the central part is composed of eight characteristic repeats, named reelin repeats, each of which is further divided by insertion of a epidermal growth factor (EGF) module into two subrepeats. In contrast, the N-terminal region shows unique 'irregular' domain architecture since it comprises three consecutive subrepeats without the intervening EGF module. Here, we determined the crystal structure of the murine reelin fragment named RX-R1 including the irregular region and the first reelin repeat at 2.0-Å resolution. The overall structure of RX-R1 has a branched Y-shaped form. Interestingly, two incomplete subrepeats cooperatively form one entire subrepeat structure, though an additional subrepeat is inserted between them. We further reveal that Arg335 of RX-R1 is crucial for binding CR-50. A possible self-association mechanism via the N-terminal region is proposed based on our results.

Photo-responsive supramolecular hyaluronic acid hydrogels for accelerated wound healing

Supramolecular hydrogels confer control over structural properties in a reversible, dynamic, and biomimetic fashion. The design of supramolecular hydrogels with an improved structural and functional recapitulation of damaged organs is important for clinical applications. For wound healing management, in particular, an effective healing process, through the modulation of epidermal growth factor (EGF) delivery using supramolecular polysaccharide hydrogels, has yet to be developed. In this study, photo-responsive supramolecular polysaccharide hydrogels were formed through host-guest interactions between azobenzene and β-cyclodextrin groups conjugated to hyaluronic acid chains. By exploiting the photoisomerization properties of azobenzene under different wavelengths, a supramolecular hydrogel featuring a dynamic spatial network crosslink density through the application of a light stimulus was obtained. Under ultra violet (UV) light, the loosened hydrogel can rapidly release EGF, thereby enhancing EGF delivery at the wound site. Based on an in vivo assessment of the healing process through a full-thickness skin defect model, the controlled EGF release from a supramolecular hydrogel exhibited superior wound healing efficiency with respect to granulation tissue formation, growth factor levels, and angiogenesis. Therefore, the proposed supramolecular hydrogels are potentially valuable as controlled delivery systems for future clinical wound healing applications.

Hair growth is promoted by BeauTop via expression of EGF and FGF‑7.

Minoxidil and finasteride have been approved to treat hair loss by the Food and Drug Administration. However, the further elucidation of treatments for hair loss, including those using Chinese herbal medicine, remains important clinically. BeauTop (BT) is a health food supplement which contains Ginseng radix, Astragali radix, Radix Angelicae sinensis, Ligustri fructus, Rehmannia glutinosa and Eclipta prostrata (Linn). Susbsequent to oral administration of BT at 0.6 g/kg/day to wax/rosin-induced alopecia in C57BL/6 mice, BT significantly induced hair growth at day 8 compared with control treatment (P<0.05). The expression levels of epidermal growth factor (EGF), and fibroblast growth factor (FGF)-7 were increased compared with control animals on day 8. In contrast, levels of FGF-5 of the BT group were reduced compared with the control on day 12. There were no effects on the expression of insulin-like growth factor 1. The results demonstrated that the mechanism of BT improving alopecia is potentially associated with modulation of EGF and FGF-7 levels. Taken together, it is suggested that BT may have a potential effect of the promotion of hair growth.

A sea urchin invivo model to evaluate Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity ofthe sea urchin embryo to anti-EMT drugs and pinpoint the sea urchin embryo as a valuable invivo model system for studying EMT and the screening of anti-EMT candidates.

Growth hormone (GH) modulation of Epidermal growth factor (EGF) signalling in human mammary epithelial cells

Growth hormone (GH) modulation of Epidermal growth factor (EGF) signalling in human mammary epithelial cells

Targeting cancer stem cell plasticity through modulation of epidermal growth factor and insulin-like growth factor receptor signaling in head and neck squamous cell cancer.

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics.

A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

Evidence is presented for the nuclear presence of a functional heteromeric complex of epidermal growth factor (EGFR), Src and the Signal Transducer and Activator of Transcription (Stat)3 proteins in pancreatic cancer cells. Stat3 remains nuclear and associated with Src or EGFR, respectively, upon the siRNA knockdown of EGFR or Src, demonstrating the resistance of the complex to the modulation of EGFR or Src alone. Significantly, chromatin immunoprecipitation (ChIP) analyses reveal the nuclear EGFR, Src and Stat3 complex is bound to the c-Myc promoter. The siRNA knockdown of EGFR or Src, or the pharmacological inhibition of Stat3 activity only marginally suppressed c-Myc expression. By contrast, the concurrent modulation of Stat3 and EGFR, or Stat3 and Src, or EGFR and Src strongly suppressed c-Myc expression, demonstrating that the novel nuclear heteromeric complex intricately regulates the c-Myc gene. The prevalence of the transcriptionally functional EGFR, Src, and Stat3 nuclear complex provides an additional and novel mechanism for supporting the pancreatic cancer phenotype and explains in part the insensitivity of pancreatic cancer cells to the inhibition of EGFR, Src or Stat3 alone.

Arkadia--beyond the TGF- pathway

Arkadia, also known as ring finger 111 (Rnf111), is an E3 ubiquitin ligase that amplifies transforming growth factor (TGF)-β family signalling through degradation of negative TGF-β signal regulators, i.e. Smad7, c-Ski and SnoN. Arkadia plays critical roles in early embryonic development through modulation of nodal signalling, as well as progression of tissue fibrosis and cancer through regulation of TGF-β signalling. Recent findings suggest that, similar to other ubiquitin ligases, including Smurf1 and 2, Arkadia regulates signalling pathways other than those of the TGF-β family. Arkadia interacts with the clathrin-adaptor 2 (AP2) complex and regulates endocytosis of certain cell surface receptors, leading to modulation of epidermal growth factor (EGF) and possibly other signalling pathways.

Mapping Surface Accessibility of the C1r/C1s Tetramer by Chemical Modification and Mass Spectrometry Provides New Insights into Assembly of the Human C1 Complex

C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.

TMEFF2: a new endogenous modulator of the CRH signaling in corticotroph cells

We have previously demonstrated that retinoic acid (RA) inhibits CRH signaling in corticotroph cells. In order to elucidate the mechanisms controlling RA action, cDNA microarray analysis was perfomed. From 87 genes regulated by RA in the corticotroph cell line AtT-20, we selected TMEFF2 for further studies. TMEFF2 is a transmembrane protein containing an altered epidermal growth factor (EGF)-like motif, two follistatin-like domains and a cytosolic tail which a putative G protein-activating motif. TMEFF2 is predominantly expressed in brain and prostate and has been implicated in cell signaling, neuronal cell survival and tumor suppression. Although TMEFF2 function is unknown, its structural domains suggest that it may have a role in the regulation of growth factor signalling. Since there is no previous evidence for an involvement of TMEFF2 in Cushing's disease or CRH signalling, we have studied the effects of TMEFF2 on the signal transduction pathway of CRH. Our results show that CRH-signaling is under the inhibitory effects of endogenous TMEFF2. TMEFF2 expression in corticotroph cells inhibits the effects of CRH on the production of intracellular cAMP, CREB and POMC transcriptional activity. Regulation of CRH activity by TMEFF2 does not require the cytoplasmic tail, while deletion of the follistatin and EGF modules abolishes the inhibitory function of TMEFF2. Moreover, a soluble secreted protein containing these three extracellular domains is sufficient for CRH signaling inhibition. TMEFF2-induced inhibition depends on serum components. Furthermore, TMEFF2 seems to regulate non-cannonical Activin/BMP-4 signaling, leading to the inhibition of the PI3K pathway (Akt) and Erk1/2 activation. Whether TMEFF2 acts upstream to Akt to regulate cAMP production and PKA activity remains to be elucidated. We found that TMEFF2 expression in human Cushing's adenoma is reduced as compared to the normal human pituitary, which may indicate that TMEFF2 acts as a tumor suppressor in these adenomas. Consistent with this hypothesis, the overexpression of TMEFF2 decreased cell proliferation in corticotroph cells. Our results also indicate a potential therapeutic use of TMEFF2 or factors that stimulate TMEFF2 activity for the treatment of corticotroph tumors in order to reduce their ACTH secretion and proliferation.

Modulation of Epidermal Growth Factor stimulated ERK phosphorylation and cell motility by inositol trisphosphate kinase

Epidermal growth factor [EGF] mediated stimulation of its receptor in endothelial cell [EC] is accompanied by phosphorylation of the EGF‐receptor [EGFR] and activation of phospholipase C‐γ, resulting in the breakdown of phosphatidylinositol(4,5)‐bisphosphate and generating inositol (1,4,5)‐trisphosphate [IP3] and diacylglycerol. IP3 thus formed can be further converted to inositol (1,3,4,5)‐tetrakisphosphate [IP4] by an enzyme called IP3‐kinase [IP3K]. In this study we have investigated the effect of modulation of intracellular IP3K activity by the use of an inhibitor, 2‐trifluoromethyl [6‐(4‐nitrobenzyl)‐purine] [IP3KI] and siRNA against IP3KB on EGF‐induced ERK‐phosphorylation and cell motility. EC cells were stimulated with EGF in presence or absence of IP3KI. Cells were lysed, and proteins separated by SDS gel electrophoresis; the western blots were probed with polyclonal antibody to designated proteins. IP3KI, at 10 μM and 20 μM concentrations inhibited both EGF‐stimulated ERK phosphorylation and motility in a dose‐dependent manner. Interestingly, at the above concentrations, IP3KI also inhibited EGFR phosphorylation at Y1173. To further establish whether any of the above effects of IP3KI can be attributed to the IP3K activity rather than the direct inhibition of EGFR kinase by IP3KI, we downregulated intracellular IP3KB with siRNA. Lipofectamine transfection of the EC with 50 pmole and 75 pmole siRNA against IP3KB was accompanied by decreased level of IP3KB [as determined by western blot], as well as inhibition of phosphorylation of both EGFR [at Y1173] and ERK1/2. Based on these data, we conclude that IP3KB, and therefore its resulting product IP4, is capable of modulating EGF stimulated response at the receptor level.

The tetralogy of Fallot‐associated G274D mutation impairs folding of the second epidermal growth factor repeat in Jagged‐1

Notch signaling controls spatial patterning and cell-fate decisions in all metazoans. Mutations in JAG1, one of the five Notch ligands in man, have been associated with Alagille syndrome and with a familial form of tetralogy of Fallot. A specific G274D mutation in the second epidermal growth factor repeat of the Jagged-1 was found to correlate with tetralogy of Fallot symptoms but not with usual Alagille syndrome phenotypes. To investigate the effects of this mutation, we studied the in vitro oxidative folding of the wild-type and mutant peptides encompassing the second epidermal growth factor. We found that the G274D mutation strongly impairs the correct folding of the epidermal growth factor module, and folding cannot be rescued by compensative mutations. The 274 position displays very low tolerance to substitution because neither the G274S nor the G274A mutants could be refolded in vitro. A sequence comparison of epidermal growth factor repeats found in human proteins revealed that the pattern displayed by the second epidermal growth factor is exclusively found in Notch ligands and that G274 is absolutely conserved within this group. We carried out a systematic and comprehensive analysis of mutations found in epidermal growth factor repeats and show that specific residue requirements for folding, structural integrity and correct post-translational processing may provide a rationale for most of the disease-associated mutations.

Identification of the C1q-binding Sites of Human C1r and C1s: A REFINED THREE-DIMENSIONAL MODEL OF THE C1 COMPLEX OF COMPLEMENT

The C1 complex of complement is assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a Ca(2+)-dependent tetramer of two modular proteases C1r and C1s. Resolution of the x-ray structure of the N-terminal CUB(1)-epidermal growth factor (EGF) C1s segment has led to a model of the C1q/C1s-C1r-C1r-C1s interaction where the C1q collagen stem binds at the C1r/C1s interface through ionic bonds involving acidic residues contributed by the C1r EGF module (Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C., and Gaboriaud, C. (2003) J. Biol. Chem. 278, 32157-32164). To identify the C1q-binding sites of C1s-C1r-C1r-C1s, a series of C1r and C1s mutants was expressed, and the C1q binding ability of the resulting tetramer variants was assessed by surface plasmon resonance. Mutations targeting the Glu(137)-Glu-Asp(139) stretch in the C1r EGF module had no effect on C1 assembly, ruling out our previous interaction model. Additional mutations targeting residues expected to participate in the Ca(2+)-binding sites of the C1r and C1s CUB modules provided evidence for high affinity C1q-binding sites contributed by the C1r CUB(1) and CUB(2) modules and lower affinity sites contributed by C1s CUB(1). All of the sites implicate acidic residues also contributing Ca(2+) ligands. C1s-C1r-C1r-C1s thus contributes six C1q-binding sites, one per C1q stem. Based on the location of these sites and available structural information, we propose a refined model of C1 assembly where the CUB(1)-EGF-CUB(2) interaction domains of C1r and C1s are entirely clustered inside C1q and interact through six binding sites with reactive lysines of the C1q stems. This mechanism is similar to that demonstrated for mannan-binding lectin (MBL)-MBL-associated serine protease and ficolin-MBL-associated serine protease complexes.

Crystal Structure of the CUB1-EGF-CUB2 Domain of Human MASP-1/3 and Identification of Its Interaction Sites with Mannan-binding Lectin and Ficolins

MASP-1 and MASP-3 are homologous proteases arising from alternative splicing of the MASP1/3 gene. They include an identical CUB(1)-EGF-CUB(2)-CCP(1)-CCP(2) module array prolonged by different serine protease domains at the C-terminal end. The x-ray structure of the CUB(1)-EGF-CUB(2) domain of human MASP-1/3, responsible for interaction of MASP-1 and -3 with their partner proteins mannan-binding lectin (MBL) and ficolins, was solved to a resolution of 2.3A(.) The structure shows a head-to-tail homodimer mainly stabilized by hydrophobic interactions between the CUB(1) module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca(2+) ion bound primarily to both EGF modules stabilizes the intra- and inter-monomer CUB(1)-EGF interfaces. Additional Ca(2+) ions are bound to each CUB(1) and CUB(2) module through six ligands contributed by Glu(49), Asp(57), Asp(102), and Ser(104) (CUB(1)) and their counterparts Glu(216), Asp(226), Asp(263), and Ser(265) (CUB(2)), plus one and two water molecules, respectively. To identify the residues involved in interaction of MASP-1 and -3 with MBL and L- and H-ficolins, 27 point mutants of human MASP-3 were generated, and their binding properties were analyzed using surface plasmon resonance spectroscopy. These mutations map two homologous binding sites contributed by modules CUB(1) and CUB(2), located in close vicinity of their Ca(2+)-binding sites and stabilized by the Ca(2+) ion. This information allows us to propose a model of the MBL-MASP-1/3 interaction, involving a major electrostatic interaction between two acidic Ca(2+) ligands of MASP-1/3 and a conserved lysine of MBL. Based on these and other data, a schematic model of a MBL.MASP complex is proposed.

Temporal changes in the expression of TGF-beta 1 and EGF in the ventral horn of the spinal cord and associated precentral gyrus in adult Rhesus monkeys subjected to cord hemisection

It is well known that some growth factors can not only rescue neurons from death, but also improve motor functions following spinal cord injury. However, their cellular distribution in situ and temporal expressions following spinal cord injury have not been determined, especially in primates. This study investigated the temporal changes in the expression of two growth factors--epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta1) in the injured motoneurons of the spinal cord and the associated precentral gyrus in adult Rhesus monkeys subjected to spinal cord hemisection. Animals were allowed to survive 7, 14, 30 and 90 days post operation (dpo). Functional recovery of the hindlimbs was assessed using Tarlov scale. The immunohistological expressions of EGF and TGF-beta1 in the ventral horn motoneurons decreased sharply at 7 dpo in the cord segments caudal to the lesion site, which was followed by an increase and a peak between 14 and 30 dpo for EGF and at 90 dpo for TGF-beta1. Changes in the expression of EGF in the precentral gyrus were similar to that in the spinal cord. No TGF-beta1 immunoreactive neurons were detected in the precentral gyrus. In the spinal segments rostral to the lesion, the expressions of EGF and TGF-beta1 peaked at 30 dpo. The mRNA of EGF was detected in both spinal motoneurons and the precentral gyrus, while that of TGF-beta1, only in the spinal motoneuons, suggesting that the spinal motoneurons themselves could synthesize both the growth factors. Partial locomotor recovery in hindlimbs was seen, especially after 14 dpo. It was concluded that a possible association existed between the modulation of EGF and TGF-beta1 and the recovery of locomotor function, and their roles differed somewhat in the neuroplasticity observed after spinal cord injury in primates.

Loss of Nidogen-1 and -2 Results in Syndactyly and Changes in Limb Development

Nidogens are two ubiquitous basement membrane proteins produced mainly by mesenchymal cells. Nidogen-mediated interactions, in particular with laminin, collagen IV, and perlecan have been considered important in the formation and maintenance of the basement membrane. However, whereas mice lacking both nidogen isoforms or carrying mutations in the high affinity nidogen-binding site upon the laminin gamma1 chain have specific basement membrane defects in certain organs, particularly in the lung, characterization of these mice has also shown that basement membrane formation per se does not need nidogens or the laminin-nidogen interaction. Limb development requires the complex interplay of numerous growth factors whose expression is dependent upon the apical ectodermal ridge. Here, we show that lack of nidogen-1 and -2 results in a specific and time-limited failure in the ectodermal basement membrane of the limb bud. The absence of this basement membrane leads to aberrant apical ectodermal ridge formation. It also causes altered distribution of growth factors, such as fibroblast growth factors and leads to a fully penetrant soft tissue syndactyly caused by the dysregulation of interdigital apoptosis. Further, in certain animals more severe changes in bone formation occur, providing evidence for the interplay between growth factors and the extracellular matrix.

Binding of Calcium to Anticoagulant Protein S: Role of the Fourth EGF Module

Protein S is an anticoagulant protein containing a Gla (enclosing gamma-carboxyglutamic acids) module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules, and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities. The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected the calcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S, involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.

Structure of a signaling-competent reelin fragment revealed by X-ray crystallography and electron tomography

The large extracellular glycoprotein reelin directs neuronal migration during brain development and plays a fundamental role in layer formation. It is composed of eight tandem repeats of an approximately 380-residue unit, termed the reelin repeat, which has a central epidermal growth factor (EGF) module flanked by two homologous subrepeats with no obvious sequence similarity to proteins of known structure. The 2.05 A crystal structure of the mouse reelin repeat 3 reveals that the subrepeat assumes a beta-jelly-roll fold with unexpected structural similarity to carbohydrate-binding domains. Despite the interruption by the EGF module, the two subdomains make direct contact, resulting in a compact overall structure. Electron micrographs of a four-domain fragment encompassing repeats 3-6, which is capable of inducing Disabled-1 phosphorylation in neurons, show a rod-like shape. Furthermore, a three-dimensional molecular envelope of the fragment obtained by single-particle tomography can be fitted with four concatenated repeat 3 atomic structures, providing the first glimpse of the structural unit for this important signaling molecule.