Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

Self-assembling multimeric integrin alpha5beta1 ligands for cell attachment and spreading.

Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin alpha5beta1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri- and tetrameric coiled coils and thus self-assembling, multimeric integrin alpha5beta1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate 'anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin alpha5beta1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin alpha5beta1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.

Solution formulation and lyophilisation of a recombinant fibronectin fragment.

The 9th-10th type III fibronectin domain pair shows promise in tissue engineering and tumour vasculature targeting. Calorimetry and structure-function analysis were used to investigate the effects of solution formulation and lyophilisation of a mutant ((9-10)FNIII-P). A single endothermic transition for (9-10)FNIII-P in solution was observed at pH<8, irrespective of addition of sucrose or PEG. The temperature at the maximum heat capacity (T(m)) and enthalpy (deltaH) of the transition increased for increasing sucrose concentrations but decreased for increasing PEG concentrations. The transition was fitted to a single two-state unfolding mechanism (in contrast to unfolding in guanidine. x HCl) and was partially reversible only at pH 4, with increasing concentrations of sucrose causing a marked fall in deltaH between scans. Circular dichroism spectra for the thermal unfolding of (9-10)FNIII-P at pH 4 showed loss of native beta-sheet structure and loss of aromatic contributions to the peak centred around 226 nm yielding an intermediate conformation, which in the presence of sucrose was more disordered. Despite a glass transition (T(g)') for (9-10)FNIII-P(aq) of -70 degrees C, primary drying at -30 degrees C did not perturb its conformation upon reconstitution or its biological activity following lyophilisation; the addition of sucrose or PEG had no influence on structure or activity. The main consideration in the formulation of (9-10)FNIII-P was therefore pH.

Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1.

An association between endometriosis and the glutathione S-transferase (GST) M1 null mutation has been reported in French and Slavic populations. We aimed to replicate this association of endometriosis in a UK population, and to test for association with the GSTT1 null mutation or the cytochrome P450 (CYP) 1A1 MspI polymorphism. We genotyped 148 women each with endometriosis (sporadic cases, n = 91; familial cases, n = 57), a population control of 95 male blood donors, and a control group of 53 women with a normal pelvis at hysterectomy. No significant differences were found between cases and controls in the frequencies of the GSTM1 and GSTT1 null mutations, or the CYP1A1 MspI polymorphism. However, the combination of the GSTM1 null genotype and the CYP1A1 MspI polymorphism was associated with a small increased risk of endometriosis, and this warrants further investigation. We also tested for linkage to the chromosome 1p13 region, to which GSTM1 has been mapped, in 52 sister-pairs with stage III-IV disease using three highly polymorphic microsatellite markers. However, there was no evidence of linkage, suggesting that this region may not be implicated in disease susceptibility.

The eighth FIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth FIII domain.

Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.

Expression of arylamine N-acetyltransferases in pre-term placentas and in human pre-implantation embryos.

Arylamine N -acetyltransferases (NATs) catalyse the acetylation from acetyl-CoA of arylamines and hydrazines. There are two human isoenzymes which show polymorphism, and both enzymes are involved in the activation and detoxification of environmental carcinogens and teratogens. The two human isoenzymes NAT1 and NAT2 show different tissue distribution, with human NAT2 being found in liver and intestine whilst human NAT1 is expressed in many tissues including erythrocytes, bladder, lymphocytes and neural tissue, as well as liver and intestine. It has been proposed that NAT1 has an endogenous role in the acetylation of the folate catabolite p -aminobenzoyl-L-glutamate (pABGlu) to produce the major urinary product, N -acetyl-pABGlu. The murine homologue of human NAT1 is known to be concentrated in the neural tube during development. We show here that human NAT1 but not human NAT2 is expressed in pre-implantation embryos at the blastocyst stage and show that NAT1 is also expressed in early human placenta at the earliest available stage, 5.5 weeks. We demonstrate that there is inter-individual variation in NAT1 expression. In view of the role of folate in protecting against neural tube defects, we propose that NAT1 is a candidate risk factor for susceptibility to neural tube defects.

Absence of a relationship between endometriosis and the N314D polymorphism of galactose-1-phosphate uridyl transferase in a UK population.

An association between the N314D polymorphism of galactose-1-phosphate uridyl transferase and endometriosis has recently been reported in a North American population. To determine whether such an association exists in the UK population, we genotyped 148 women with sporadic (n = 91) or familial (n = 57) endometriosis, a control population of 95 male blood donors and a control group of 53 women with a normal pelvis at hysterectomy. Heterozygosity for the polymorphism was found in 14.9% (22/148) of affected women, 13.7% (13/95) of male blood donors and 11.3% (6/53) of women with a normal pelvis. There was no statistically significant difference in the frequency of the polymorphism between cases and controls in the UK population, even when the cases were divided into groups of moderate-severe disease, sporadic cases or familial cases. We conclude that the galactose-1-phosphate uridyl transferase N314D polymorphism is unlikely to be associated with endometriosis in the UK population.

Differential activation of focal adhesion kinase, Rho and Rac by the ninth and tenth FIII domains of fibronectin.

Fibronectins are widely expressed extracellular matrix ligands that are essential for many biological processes. Fibronectin-induced signaling pathways are elicited in diverse cell types when specific integrin receptors bind to the ninth and tenth FIII domains, FIII9-10. Integrin-mediated signal transduction involves activation of signaling pathways of the growth factor-dependent Ras-related small GTP-binding proteins Rho and Rac, and phosphorylation of focal adhesion kinase. We have dissected the requirement of FIII9 and FIII10 for Rho and Rac activity and phosphorylation of focal adhesion kinase in BHK fibroblasts and Swiss 3T3 cells. We demonstrate that FIII10 supports cell attachment but does not induce phosphorylation of focal adhesion kinase. In Swiss 3T3 cells, growth factor-independent phosphorylation of focal adhesion kinase and downstream adhesion events are dependent upon the presence of FIII9 in the intact FIII9-10 pair, whereas FIII10-mediated focal adhesion kinase phosphorylation requires a synergistic signal from growth factors. Furthermore, FIII10 is able to elicit cellular responses mediated by Rho, but not Rac, whereas FIII9-10 can elicit both Rho- and Rac-mediated responses. We propose that activation of specific integrin subunits by the FIII10 and FIII9-10 ligands elicits distinct signaling events. This may represent a general molecular mechanism for activation of receptor-specific signaling pathways by a multi-domain ligand.

Tenascin is differentially expressed in endometrium and endometriosis.

Endometriosis is characterized by the presence of functional endometrial tissue outside the uterine cavity, most commonly on the ovary and peritoneum. The aetiology of endometriosis is not understood, although the adhesion of endometrial cells to the extracellular matrix (ECM) would be expected to play a central role in its pathogenesis. The expression of ECM molecules in endometrium and in endometriosis has been investigated using immunohistochemistry and western blotting techniques. The ECM components collagen IV, laminin, vitronectin, and fibronectin had a similar pattern of expression throughout the menstrual cycle in endometrium and endometriosis. Expression of tenascin was elevated in the stroma of the functionalis region of the endometrium during the proliferative stage of the menstrual cycle and in endometriosis. Tenascin expression in endometriosis was not modulated according to the stage of the menstrual cycle. It is concluded that expression of tenascin is strictly regulated in endometrium and may be important in endometrial regeneration and in the pathogenesis of endometriosis.

Gestational regulation of granulocyte-colony stimulating factor receptor expression in the human placenta.

A number of cytokines and their receptors are abundantly expressed at the materno-fetal interface and are thought to have a function in the regulation of placentation. Granulocyte-colony stimulating factor (G-CSF) is expressed by stromal cells in both placental tissue and maternal decidua throughout placentation. In this study, we examined the expression of placental G-CSF receptor (G-CSFR) mRNA and protein throughout gestation by ribonuclease protection assays, Western blotting, and immunohistochemistry. The major placental form of G-CSFR mRNA, corresponding to a membrane-bound form of the protein, was present in first-trimester placental tissues; levels decreased in second- and were highest in third-trimester placental tissues. Two placental G-CSFR molecules, 120 kDa and 150 kDa, were detected in first- and third-, but not second-, trimester tissues. The level of the 150-kDa G-CSFR was greater in the third- than in first-trimester samples. These differences were irrespective of whether or not the patients had received prostaglandin E1 analogues, prostaglandin E1 analogues and oxytocin, oxytocin alone, or mifepristone before labor. We demonstrated by immunohistochemistry that interstitial cytotrophoblast in first- and second-trimester decidual tissue and cytotrophoblast in term fetal membranes express G-CSFR. These data demonstrate that the expression of specific forms of placental G-CSFR is strictly cell type- and developmental stage-specific, and they suggest that G-CSFR may be important in decidual invasion of cytotrophoblast and in trophoblast function during placentation.

Placental expression of arylamine N-acetyltransferases: evidence for linkage disequilibrium between NAT1*10 and NAT2*4 alleles of the two human arylamine N-acetyltransferase loci NAT1 and NAT2.

The study of placental xenobiotic metabolism is important for the determination of foetal exposure to environmental chemicals as placental metabolism influences the nature of chemicals reaching the foetus from its mother's blood. Arylamine N-acetyltransferases are drug metabolizing enzymes which N-acetylate hydrazines and arylamines, including carcinogenic arylamines and sulphonamide drugs. The two human arylamine N-acetyltransferase isoenzymes, NAT1 and NAT2, are encoded at multi-allelic loci. Here, we have determined N-acetyltransferase (NAT) activity in term placentas from normal, uncomplicated pregnancies. Both NAT1 and NAT2 enzyme activities were detectable. Placental NAT1 activity was at least 1000 fold greater than NAT2 activity. There was a 6 fold inter-placental variation in NAT1 activity. Mean placental NAT1 specific activity was 1.42 nmoles para-aminobenzoic acid N-acetylated.min-1.mg protein-1, which is comparable to NAT1 specific activities which have been measured in adult tissues. The NAT1, but not the NAT2, protein was detectable in placentas by Western blotting. Maternal and foetal NAT genotypes were determined from placenta, using placental blood clots and cord blood respectively, allowing NAT haplotype determination. There appeared to be linkage disequilbrium between NAT1* and NAT2* alleles such that the combination NAT1*10/NAT2*4 was found 3.5 times more frequently than would be expected.

Endometriosis in monozygotic twins.

OBJECTIVE: To describe the occurrence of endometriosis in monozygotic twins. DESIGN: Postal questionnaire plus confirmation of disease status. SETTING: Twins were recruited via the American Endometriosis Association and the National Endometriosis Society of Great Britain and via British gynecologists. RESULT(S): Fourteen twin pairs were concordant for endometriosis, and two were discordant. Nine pairs of twins had moderate-severe endometriosis. CONCLUSION(S): These findings contribute to the growing body of literature that suggests endometriosis has a genetic basis.

Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit.

The high affinity interaction of integrin alpha5beta1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on alpha5beta1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-alpha5 mAbs (P1D6 and JBS5) to alpha5beta1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to alpha5beta1. In contrast, binding of the anti-beta1 mAb P4C10 to alpha5beta1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with alpha5beta1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the alpha5 subunit. Our results indicate that the synergy region is recognized primarily by the alpha5 subunit (in particular by its NH2-terminal repeats) but that the beta1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.

Structural requirements for biological activity of the ninth and tenth FIII domains of human fibronectin.

The ninth and tenth type III domains of fibronectin each contain specific cell binding sequences, RGD in FIII10 and PHSRN in FIII9, that act synergistically in mediating cell adhesion. We investigated the relationship between domain-domain orientation and synergistic adhesive activity of the FIII9 and FIII10 pair of domains. The interdomain interaction of the FIII9-10 pair was perturbed by introduction of short flexible linkers between the FIII9 and FIII10 domains. Incremental extensions of the interdomain link between FIII9 and FIII10 reduced the initial cell attachment, but had a much more pronounced effect on the downstream cell adhesion events of spreading and phosphorylation of focal adhesion kinase. The extent of disruption of cell adhesion depended upon the length of the interdomain linker. Nuclear magnetic resonance spectroscopy of the wild type and mutant FIII9-10 proteins demonstrated that the structure of the RGD-containing loop is unaffected by domain-domain interactions. We conclude that integrin-mediated cell adhesion to the central cell binding domain of fibronectin depends not only upon specific interaction sites, but also on the relative orientation of these sites. These data have implications for the molecular mechanisms by which integrin-ligand interactions are achieved.

Module-module interactions in the cell binding region of fibronectin: stability, flexibility and specificity.

The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-dependent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein-protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and 15N relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein-protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces.

Expression of human myometrial G alpha s messenger ribonucleic acid transcript during pregnancy and labour: involvement of alternative splicing pathways.

We have shown previously that expression of 46 and 54 kDa human myometrial G alpha s protein isoforms is increased during gestation and then subsequently decreased during labour. These proteins appear to be coded for by G alpha s-Small (with a serine residue at position 72) and G alpha s-Large (with a serine residue at position 87) mRNA splice variants respectively. In the study presented here we have used a G alpha s cDNA template to generate [32P]cytidine cRNA riboprobes for use in RNase protection assays, so as to measure total myometrial G alpha s mRNA levels in relation to the pattern of expression of G alpha s mRNA splice variants during pregnancy and labour. We report that total levels of human myometrial G alpha s mRNA remain similar in non-pregnant and pregnant women but are substantially reduced during parturition. Our data also provide strong evidence that alternative splicing of G alpha s precursor mRNA has a primary role in regulating expression of G alpha s protein isoforms during pregnancy and labour. The inclusion of an additional serine codon in G alpha s mRNAs during pregnancy involves a switch in alternative splicing pathways. We speculate that this switch may be due to a change in specificity of splicing factors that are modulated during pregnancy and labour.

Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells.

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

Temporal and spatial regulation of expression of heparin-binding epidermal growth factor-like growth factor in the human endometrium: a possible role in blastocyst implantation.

The function of the endometrium in the implantation of the blastocyst depends on the regulated, cyclical regeneration of endometrial tissue and the expression of a receptive phenotype in response to steroid hormones. Experiments using animal and models suggest that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is important for endometrial receptivity, and that it may directly mediate blastocyst implantation We have investigated the expression of HB-EGF mRNA and protein in pregnant and nonpregnant human endometrium and placenta. Our data demonstrate that HB-EGF mRNA expression is low in the endometrium during the proliferative stage of the menstrual cycle and increases in the secretory stage, with highest expression immediately prior to the implantation window (day 19-21), after which levels decrease. Immunohistochemical detection of HB-EGF shows that it is present in the stroma of proliferative stage endometrium and that it is localized to the apical surface of the luminal epithelium of midsecretory stage endometrium. Levels of HB-EGF mRNA are low in pregnant endometrium and high in placental tissues at an early stage of development. Our data suggest that expression of human endometrial HB-EGF coincides with the expression of a receptive phenotype, and that H-EGF may have an important function in the implantation of the human blastocyst and early placental development.

Acetylation of arylamines by the placenta.

The N-acetylation of arylamines and hydrazines used as drugs may alter their pharmacological or toxicological activity. Arylamine N-acetyltransferase (NATs) are involved in drug metabolism, as they catalyse the N-acetylation of arylamine and mono-substituted hydrazine substrates. Placental metabolism regulates the nature of the chemicals which reach the developing fetus. The study of drug metabolism during pregnancy is important in determining the effect on the fetus of drugs administered to the mother and the maternal drug dose required, important if the treatment is to be effective. There are two forms of NAT in humans, NAT1 and NAT2, which are encoded at multi-allelic loci. There is inter-individual variation in both NAT1 and NAT2 activity, which has implications in drug dosage. Using a combination of enzyme activity measurements and Western blotting, this study has characterised the arylamine N-acetylation capabilities of placenta and cord blood. NAT1 activity in placenta and cord blood demonstrated inter-individual variation and the variation was in the range expected for adult NAT1 activity. The genotypes of both NAT1* and NAT2* were determined using DNA prepared using placental blood clots (maternal DNA) and cord blood (fetal DNA). The results indicate that placental NAT activity is an important factor when considering N-acetylation during pregnancy.