PubMed · 4259744
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K S Lee. 1972. Placental localization.. https://pubmed.ncbi.nlm.nih.gov/4259744/
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Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC1 domain contains multiple submodules with homology to known adhesive molecules including fibronectin type III-like repeats and the A domain of von Willebrand factor. In this study, we produced the entire NC1 domain of human type VII collagen in the stably transfected human kidney 293 cell clones and purified large quantities of the recombinant NC1 protein from serum-free culture media. The recombinant NC1 formed interchain disulfide-bonded dimers and trimers and was N-linked glycosylated. Tunicamycin inhibited the cellular secretion of NC1, suggesting that N-linked glycosylation may play a role in NC1 secretion. The recombinant NC1 was indistinguishable from the authentic NC1 obtained from human amnions or WISH cells with respect to N-linked sugar content, electrophoretic mobility, rotary shadow imaging, and binding affinity to type IV collagen. Purified recombinant NC1, like authentic NC1, also bound specifically to fibronectin, collagen type I, and a laminin 5/6 complex. Both monomeric and trimeric forms of NC1 exhibited equal affinity for these extracellular matrix components, suggesting that the individual arms of NC1 can function independently. The multiple interactions of NC1 with other extracellular matrix components may support epidermal-dermal adhesion.
A procedure for intraamniotic ethyl-docosahexaenoate (Et-DHA) administration was used to restore the docosahexaenoic acid (DHA; 22:6 n-3) levels in n-3-deficient fetal rats. The state of deficiency, characterized by a 34% and 60% decrease in DHA content of fetal brain and liver, respectively, was attained by feeding the pregnant dams from day 8 and up to 20 days gestation, with an n-3 linolenic acid-deprived diet. After a single intraamniotic administration of Et-DHA on day 18 or 19, a rapid increase in both fetal brain and liver DHA was achieved. This increase was accompanied by a decrease in the docosapentaenoic acid (DPA; 22:5 n-6) level. After 48 hr following Et-DHA administration, the major phospholipids (PLs) phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), together accounting for more than 90% of total lipid phosphorus in sunflower oil (SFO)-treated animals, regained the DHA content to levels similar to control animals in both fetal brain and liver tissues. Unlike brain, however, most of the DHA content in liver PLs was restored by 24 hr, suggesting that the fetal liver may have a higher metabolic turnover. The DHA/DPA ratio was used to assess the degree of DHA correction. Fetal brain PS, PC, and PE ratios following Et-DHA administration grew steadily over a period of 48 hr but reached only approximately 60% of the control levels. Liver PS regained a value similar to the control, while those of PC and PE were 33% and 46% lower than the controls, respectively. Alterations in the PL polar head-group composition were observed following the dietary manipulations and Et-DHA administration. Although the intraamniotic injection is an invasive approach, the ability to rapidly enhance DHA acylation during intrauterine life may hold potential clinical value whenever an indication for DHA deficiency exists.
Cell migration involves the integrins, their extracellular matrix ligands, and pericellular proteolytic enzyme systems. We have studied the role of plasminogen activator inhibitor-1 (PAI-1) in cell migration, using human amnion WISH cells and human epidermoid carcinoma HEp-2 cells in an assay measuring migration from microcarrier beads and a modified Boyden-chamber assay. Active, but not latent or reactive center-cleaved, PAI-1 inhibited migration. A PAI-1 mutant without ability to inhibit plasminogen activation was as active as wild-type PAI-1 as a migration inhibitor, showing that inhibition of plasminogen activation was not involved. PAI-1 specifically interfered with intergrin- and vitronectin-mediated migration: Migration onto vitronectin-coated but not onto fibronectin-coated surfaces was inhibited by PAI-1, a cyclic RGD peptide inhibited migration, and both cell lines expressed vitronectin-binding alpha v-integrins. In addition, active PAI-1, but not latent or reactive center-cleaved PAI-1, inhibited vitronectin binding to integrins in an in vitro binding assay, without affecting binding of fibronectin. Monoclonal antibodies against the urokinase receptor, another vitronectin binding protein, did not affect cell migration in the beads assay, while some inhibitory effect was observed in the Boyden-chamber assay. We conclude that PAI-1, independently of its role as a proteinase inhibitor, inhibits cell migration by competing for vitronectin binding to integrins, while the interference of PAI-1 with binding of vitronectin to the urokinase receptor may play a secondary role. These data define a novel function for the serpin PAI-1, enabling it to regulate cell migration over vitronectin-rich extracellular matrix in the body.