Mon-Li Chu

Modulation of procollagen gene expression by retinoids. Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures.

Recent clinical observations have suggested that retinoids, which are in frequent use in dermatology, can affect the connective tissue metabolism in skin and other tissues. In this study, we examined the effects of several retinoids on the metabolism of collagen by human skin fibroblasts in culture. Incubation of cultured fibroblasts with all-trans-retinoic acid or 13-cis-retinoic acid, in 10(-5) M or higher concentrations, markedly reduced the procollagen production, as measured by synthesis of radioactive hydroxyproline. The effect was selective in that little, if any, inhibition was noted in the incorporation of [3H]leucine into the noncollagenous proteins, when the cells were incubated with the retinoids in 10(-5) M concentration. Similar reduction in procollagen production was noted with retinol and retinal, whereas an aromatic analogue of retinoic acid ethyl ester (RO-10-9359) resulted in a slight increase in procollagen production in these cultures. The reduction in procollagen production by all-trans-retinoic acid was accompanied by a similar reduction in pro alpha 2(I) of type I procollagen specific messenger RNA (mRNA), as detected by dot blot and Northern blot hybridizations. Hybridizations with human fibronectin and beta-actin specific DNA probes indicated that the levels of the corresponding mRNAs were not affected by the retinoids, further suggesting selectivity in the inhibition of procollagen gene expression. Further control experiments indicated that all-trans-retinoic acid, under the culture conditions employed, did not affect the posttranslational hydroxylation of prolyl residues, the mannosylation of newly synthesized procollagen, the specific radioactivity of the intracellular prolyltransfer RNA pool, or DNA replication. All-trans-retinoic acid also elicited a reduction in trypsin-activatable collagenase, but not in the activity of prolyl hydroxylase or an elastaselike neutral protease in the fibroblast cultures. Incubation of three fibroblast lines established from human keloids with all-trans-retinoic acid or 13-cis-retinoic acid also resulted in a marked reduction in procollagen production. The results, therefore, suggest that further development of retinoids might provide a novel means of modulating collagen gene expression in patients with various diseases affecting the connective tissues.

Ehlers-Danlos syndrome type IV: cosegregation of the phenotype to a COL3A1 allele of type III procollagen

SummaryEhlers-Danlos syndrome (EDS) type IV is a rare and catastrophic genetic disorder of the connective tissue. Individuals from two families with this disorder were studied for a restriction fragment length polymorphism (RFLP) associated with the COL3A1 gene. Our results suggested cosegregation of the EDS type IV phenotype with a COL3A1 RFLP allele. Biochemical studies in cultured skin fibroblasts indicated the presence of different mutations affecting the stability and secretion of the proα1(III) chains of type III procollagen in the two families, thus suggesting that EDS type IV is biochemically heterogeneous. Our data demonstrated the feasibility of molecular diagnosis in this condition using COL3A1 gene related RFLPs.

Isolation and Characterization of the Human Fibrillar Collagen Genes

In order to elucidate some of the mechanisms leading to the pathological expression of the human fibrillar collagens, as well as to understand the evolution of these loci, specific cDNA and genomic clones have been isolated. The primary structure of the COOH-terminal propeptide of the four collagen chains and either part or the entire exon/intron arrangement of the genes have been determined. Interspecies and pairwise comparison revealed that the four loci have evolved at slightly different rates, maintaining, however, remarkably similar exon/intron arrangement. The fibrillar genes, albeit sharing the same elaborate structure, exhibit different sizes that correlate with the average length of their intron sequences, possibly because of their different chromosomal origin.

Mutations in Osteogenesis Imperfecta Leading to the Synthesis of Abnormal Type I Procollagens

Osteogenesis imperfecta or “brittle bone disease” is of interest for several reasons. From the point of view of the physician or of medicine in general, it is of great interest to establish the molecular basis of this disease, or more correctly, group of diseases (for reviews see refs. 1-5). At the phenotypic level, the diseases show a broad spectrum of heterogeneity. Brittle bones are the hallmark of the disease and in the most severe forms, bones are so brittle that death occurs in utero. In the mildest forms, individuals who avoid trauma have minimal disability. Also, in some individuals the disease involves the teeth, skin, and sclerae of the eyes. In others it is confined to the major bony structures. It would obviously be extremely helpful in dealing with patients with these conditions to understand the molecular basis of the diseases and thereby to explain the large heterogeneity in the clinical manifestations. In addition, defining the molecular defects should make it possible to develop DNA tests for the prenatal diagnosis of the severe forms of osteogenesis imperfecta. Hence, at a practical level, it should be possible to diminish greatly the number of new cases. From the point of view of general biology, osteogenesis imperfecta is of interest because it presents a series of highly informative “experiments of Nature.” It is now established that many forms of osteogenesis imperfecta are produced by mutations in the genes for type I procollagen (see refs. 1, 3, and 5). The specific kinds of mutations that are found indicate directly the special vulnerabilities of the genes to mutations. Because the amino acid sequence of the protein is highly repetitive, the coding sequences of the genes are highly repetitive. Experiments in bacteria and other organisms demonstrate that repetitive DNA sequences are prone to undergo extensive recombination during either meiosis or mitosis. Therefore the mutations found in collagen

Connective tissue biochemistry of neurofibromas.

Neurofibromatosis is a complex neurocutaneous syndrome inherited as an autosoma1 dominant trait with high penetrance of the mutated gene (for reviews, see References 1-3). Approximately half of the cases with neurofibromatosis appear to represent new mutations, and the mutation frequency of the affected gene has been estimated to be as high 4.3 Y 10-5.4*5 The overall frequency of neurofibromatosis in the general population has been estimated to be 20-40 per lOO,OOO.. Thus, neurofibromatosis is probably the most common neurocutaneous single-gene disorder. The diagnosis of neurofibromatosis is based on clinical findings, positive family history, and associated features. The cutaneous lesions of neurofibromatosis include neurofibromas, dermal or subcutaneous tumors of varying sizes, and cafk-au-lait spots, pigmented cutaneous macules of varying sizes and The diagnosis of neurofibromatosis can be based on the following criteria:s4s9

Presence of Translatable mRNA for Proα2(I) Chains in Fibroblasts from a Patient with Osteogenesis Imperfecta whose Type I Collagen does not Contain α2(I) Chains

Abstract RNA was extracted from the cultured fibroblasts from a patient with osteogenesis imperfecta previously shown to have type I collagen lacking α2(I) chains. When the RNA was examined in a cell-free translation system from reticulocytes, the translation products included both proα1(I) and proα2(I) chains. When the poly(A)-enriched polysomal RNA was examined by blot hybridization with cDNAs for proα1(I) and proα2(I) chains, mRNAs for both proα1(I) and proα2(I) were seen. The ratio of mRNAs for proα1(I) to mRNAs for proα2(I) was about the same in the patients fibroblasts as in control fibroblasts. The results suggested that the absence of proα2(I) chains in the type I pro-collagen from this patient is probably explained by a mutation which alters the structure of proα2(I) chains and thereby prevents incorporation of the proα2(I) chains into triple-helical procollagen.

Electronmicroscopic Localization of Deletions in the Human Pro-α2(I) Collagen Genea

A heterogeneous array of molecular defects involving type I procollagen has recently been identified in a number of patients with osteogenesis imperfecta (01): These molecular defects have largely been uncovered by direct analysis of the structure and ratio of synthesis of procollagen by cultured fibroblasts. In some patients deletions have been found in the central collagen domain of the pro-a2(1) chain?4 Thus far the unusual complex structure of the human collagen genes: as well as the unavailability of full-length cDNAs, precluded the elucidation of the precise localization of deletions in the triple-helical domain of the pro-a2(I) chain. A variant with a perinatal lethal form of 01 was found to be a compound heterozygote, in which two unrelated mutations in the two alleles for pro-a2(I) were present. One allele was nonfunctional, and the mutation in the other allele produced a shortened chain. Based on the relative mobility in SDS-containing polyacrylamide gels, fragment a2(I)-CB3,5* was shortened by about 20 amino acid residues. Another variant, with a mild form of 01, showed a heterozygous defect for the synthesis of shortened pro-a2(I) chain^.^ The abnormal pro-a2(I) chains were shortened by about 30 amino acids in fragment a2(I)-CB4, containing amino acid residues 7 to 327. Extensive Southern blot analysis on agarose gels of both variants DNA yielded no data relevant to the nature or locality of the proposed gene defects. Here, electronmicroscopic R-loop analysis has been employed to determine the extent and locality of the mutations in both variants. Poly(A)-RNA were isolated from the variants fibroblasts and hybridized to the appropriate human pro-a2(I) genomic clones. R-loop structures obtained with the variants poly(A)-RNA and with control fibroblast poly(A)-RNA were compared. An abnormal distribution of exons and introns was observed in R-loops formed between the poly(A)-RNA from the mutant with a lethal form of 01 and the human