Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex

During steady state lymphopoiesis in the postnatal thymus, migration of precursors outward from the deep cortex toward the capsule is required for normal differentiation. Such migration requires, at a minimum, expression of adhesive receptors on the migrating lymphoid cells, as well as a stable matrix of their ligands persisting throughout the region of migration. In this study, we address the nature of this adhesive matrix. Although some precursor stages bound efficiently to extracellular matrix ligands, a specific requirement for the cell surface ligand VCAM-1 was also found. In situ analysis revealed that early precursors are found in intimate contact with a matrix formed by stromal cells in the cortex, a proportion of which expresses VCAM-1. In vivo administration of an anti-VCAM-1 Ab resulted in decreased thymic size and altered distribution of early precursors within the cortex. These results indicate that precursors migrating outward through the cortex may use a cellular, rather than extracellular, matrix for adhesion, and suggest that the VCAM-1(+) subset of cortical stroma may play a crucial role in supporting the migration of early precursors in the steady state thymus.     

Single base mutation in the type III procollagen gene that converts the codon for glycine 883 to aspartate in a mild variant of Ehlers-Danlos syndrome IV

Experiments were carried out to test the hypothesis that a 19-year-old proband with a mild variant of Ehlers-Danlos syndrome type IV had a mutation in the gene for type III procollagen. cDNA and genomic DNA were analyzed by using the polymerase chain reaction and cloning of the products into M13 filamentous phage. A mutation was found that converted the codon for glycine 883 of the triple-helical domain in one allele for type III procollagen to a codon for aspartate. The polymerase chain reaction introduced a few artifactual single base substitutions. Also, it was difficult to distinguish copies from the two alleles in many of the M13 clones. Therefore, several different strategies and analyses of about 50,000 nucleotide sequences in a series of clones were used to demonstrate that the mutation in the codon for glycine 883 was the only mutation in coding sequences for the triple-helical domain of type III procollagen that could have contributed to the phenotype. The same mutation in the codon for glycine 883 in one allele for type III procollagen was found in the proband's 52-year-old father who also had a mild variant of Ehlers-Danlos syndrome type IV. The type III procollagen synthesized by the proband's fibroblasts was analyzed by polyacrylamide gel electrophoresis. Less type III procollagen was secreted by the proband's fibroblasts than by control fibroblasts. Also, the thermal stability of the type III procollagen synthesized by the proband's fibroblasts was lower than the thermal stability of normal type III procollagen as assayed by brief protease digestion. The results, therefore, demonstrated that the single base mutation that converted the codon of glycine 883 to a codon for aspartate destabilized the entire triple helix of type III procollagen and probably accounted for the mild phenotype of Ehlers-Danlos syndrome type IV seen in the proband and her father.     

Papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases

Papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved 'papilin cassette' of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine-rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N-proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis.     

Potential use of stem cells from bone marrow to repair the extracellular matrix and the central nervous system

A subset of stem-like cells from bone marrow that are referred to as marrow stromal cells (MSCs) have been shown to be capable of differentiating into osteoblasts, chondrocytes, adipocytes, myocytes, astrocytes and perhaps neurons. Recently, conditions have been developed where human MSCs can be expanded almost without limit in culture without apparently losing their multipotentiality for differentiation. The cells appear to be potentially useful for the repair of extracellular matrix and the central nervous system.     

Copolymerization of pNcollagen III and collagen I. pNcollagen III decreases the rate of incorporation of collagen I into fibrils, the amount of collagen I incorporated, and the diameter of the fibrils formed

Previous observations suggested that pNcollagen III, the partially processed form of type III procollagen, coats fibrils of collagen I and thereby helps regulate the diameter of fibrils formed by collagen I. The previous observations, however, did not exclude the possibility that pNcollagen III was deposited on preformed collagen I fibrils after the fibrils were assembled. Here, mixtures of pNcollagen III and collagen I were generated simultaneously by enzymatic cleavage of precursor forms of the proteins. The results demonstrated that pNcollagen III forms true copolymers with collagen I. The presence of pNcollagen III both inhibited the rate at which collagen I assembled into fibrils and decreased the amount of collagen I incorporated into fibrils at steady-state equilibrium. In addition, the results demonstrated that copolymerization of pNcollagen III with collagen I generated fibrils that were thinner than fibrils generated under the same conditions from collagen I alone. Increasing the initial molar ratio of pNcollagen III to collagen I in the solution-phase increased the amount of pNcollagen III copolymerizing with collagen I and progressively decreased the diameter of the fibrils. Therefore, the copolymers were heterogeneous in that the stoichiometry of the two monomers in the fibrils varied. The results are consistent with a model in which pNcollagen III can regulate the diameter of collagen I fibrils by coating the surface of the fibrils and thereby allow tip growth but not lateral growth of the fibrils.     

High levels of expression of a minigene version of the human pro alpha 1 (I) collagen gene in stably transfected mouse fibroblasts. Effects of deleting putative regulatory sequences in the first intron.

A minigene version of the human gene for the pro alpha(I) chain of type I procollagen (COL1A1) was prepared that contained -2.3 kilobases of the 5'-flanking sequence, the first 5 exons and introns, the last 6 exons and introns, and about 2 kilobases of the 3'-flanking sequence. The gene was then used for stable transfection experiments with mouse NIH 3T3 fibroblasts. Because the products of the minigene were shorter, it was possible to compare expression of the minigene with expression of the endogenous pro alpha 1 (I) gene by Northern and Western blot analyses. The results demonstrated that the construct contained enough of the gene to obtain high levels of expression in many of the stably transfected cells. Since previous observations suggested that the first intron of the pro alpha 1 (I) gene contained important cis-regulatory elements, two versions of the minigene were prepared in which most of the first intron was deleted. Comparison of expression of the minigene with expression of two deleted versions of the same gene established that 85% of the total sequences in the first intron are not essential for high levels of expression of the gene in stably transfected mouse fibroblasts.     

Collagen fibrils in vitro grow from pointed tips in the C- to N-terminal direction.

Growth of collagen fibrils was examined in a system in which collagen monomers are generated by specific enzymic cleavage of type IpCcollagen with procollagen C-proteinase. Fibrils formed at 37 degrees C had highly tapered and symmetrical pointed tips. The pattern of cross-striations in the pointed tips indicated that all the molecules were oriented so that the N-termini were directed towards the tip. At 29 degrees C and 32 degrees C, the fibrils formed were thicker. One end of fibrils formed at 29 degrees C was blunt, and the other was pointed. Growth of the fibrils was exclusively from pointed tips. Occasionally a spear-like projection appeared at a blunted end. The spear-like projection then became a new pointed tip for growth in the opposite direction. The results suggested a model for fibril growth with at least three distinct binding sites for monomers. In the model, the pointed tip is the site with the highest affinity for the binding of monomers and most probably defines the critical concentration for fibril assembly. The main shaft of the fibril is a site with very low affinity for binding. The blunted end defines a low-affinity binding site where monomers can bind in opposite orientation to produce growth from a new pointed end.     

A heterozygous defect for structurally altered pro-alpha 2 chain of type I procollagen in a mild variant of osteogenesis imperfecta. The altered structure decreases the thermal stability of procollagen and makes it resistant to procollagen N-proteinase

Cultured skin fibroblasts from a proband with an autosomal dominant variant of osteogenesis inperfecta were found to synthesize approximately equal amounts of normal pro-alpha 2(I) chains of type I procollagen and pro-alpha 2(I) chains which migrated more rapidly when examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The structural alteration was present in alpha 2(I)-CB4, a cyanogen bromide fragment containing amino acid residues 7-327 of the alpha 2 chain, and it appeared to be a deletion of about 30 amino acids. The pro-alpha 2(I) chains with the apparent deletion associated with normal pro-alpha 1(I) chains synthesized by the same fibroblasts and formed triple-helical type I procollagen. The presence of the altered pro-alpha 2 chains in trimers of procollagen had two consequences in terms of the physical properties of the molecule. One was to decrease the thermal stability of the protein as judged by resistance to proteolysis at 37 degrees C and by the helix to coil transition as assayed by circular dichroism. The second consequence was to make type I procollagen containing the shortened pro-alpha 2(I) chains resistant to digestion by procollagen N-proteinase. The simplest explanation for the data is that the apparent deletion in half the pro-alpha 2(I) chains produced a partial unfolding of the N-terminal region of type I procollagen which prevented processing of the protein by procollagen N-proteinase.     

Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases

A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.     

Synthesis of a shortened pro-alpha 2(I) chain and decreased synthesis of pro-alpha 2(I) chains in a proband with osteogenesis imperfecta

Synthesis of type I procollagen was examined in skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta. The fibroblasts synthesized shortened pro-alpha 2(I) chains and these shortened chains accounted for all the pro-alpha 2(I) chains synthesized by the cells. In addition, there was a decrease in the relative rate of synthesis of pro-alpha 2(I) chains. Fragmentation of the shortened pro-alpha 2(I) chains with vertebrate collagenase and cyanogen bromide demonstrated that the shortening was in alpha 2(I)-CB3,5A, a fragment from about the middle of the chain containing amino acid residues 361 to 775. Based on the relative mobility in electrophoretic gels, the shortening was about 20 amino acid residues. The decreased synthesis of pro-alpha 2(I) chains was demonstrated by an increase in the ratio for the rates of synthesis of pro-alpha 1(I):pro-alpha 2(I) chains. It was associated with an increase in the ratio of mRNAs for pro-alpha 1(I):pro-alpha 2(I) in the cells. Fibroblasts from the father also demonstrated a decreased synthesis of pro-alpha 2(I) chains as reflected by an increase in the ratio of newly synthesized pro-alpha 1(I):pro-alpha 2(I) chains. No shortened pro-alpha 2(I) chains were seen in fibroblasts from either the father or the mother. The observations suggested that the proband inherited a nonfunctioning pro-alpha 2(I) gene from her father and that the gene for the shortened pro-alpha 2(I) chain probably arose from a sporadic mutation.