Biomedical subjects

G J Moffat

Publications and source records attributed to G J Moffat.

4 recordsLinked to original sources

PubMed1992

Regulation of C4b-binding protein gene expression by the acute-phase mediators tumor necrosis factor-alpha, interleukin-6, and interleukin-1.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. During an acute-phase response, we have shown that hepatic levels of murine C4BP mRNA are elevated 2.5-fold while rat liver C4BP gene expression exhibits a 4-fold induction. Furthermore, a survey of different mouse tissues showed that during acute inflammation C4BP gene expression was confined to the liver. To gain a better understanding of the acute-phase regulation of C4BP gene expression we utilized the rat hepatoma cell line FAO in which tumor necrosis factor-alpha (TNF-alpha) produced a 2.7-fold induction of C4BP mRNA levels. In the absence of TNF-alpha, interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) had little effect on C4BP gene expression but when all three cytokines were used together a synergistic 4-fold induction of C4BP mRNA levels was observed. In contrast the synthetic glucocorticoid dexamethasone inhibited TNF-alpha-induced C4BP gene expression. Cycloheximide-mediated inhibition of inducible C4BP gene expression demonstrated the requirement for ongoing protein synthesis. Rapid induction of C4BP mRNA levels by TNF-alpha and IL-6 (within 1 h) and the observation that stimulation was inhibited by actinomycin D provided evidence that regulation of C4BP gene expression during the acute-phase response is regulated at the transcriptional level. Isolation of a genomic clone extending into the 5' regulatory region of the rat C4BP gene enabled us to identify the major transcriptional start site and putative response elements through which TNF-alpha, IL-6, IL-1 alpha, and dexamethasone may exert their effects on C4BP gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Reaction↗

PubMed1992

Complete structure of the murine C4b-binding protein gene and regulation of its expression by dexamethasone.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. A murine genomic library was screened, and five clones were selected that covered the remaining four exons in the 5'-region of the C4BP gene. Together with previous work (Barnum, S. R., Kristensen, T., Chaplin, D. D., Seldin, M. F., and Tack, B. F. (1989) Biochemistry 28, 8312-8317), the entire C4BP gene has now been shown to be 23 kilobases (kb) long and comprised of 10 exons ranging in size from 86 to 442 base pairs (bp). Primer extension analysis revealed the major transcription start site to be 46 bp upstream of the published cDNA start site. Northern blot analysis of RNA isolated from several mouse tissues demonstrated that the C4BP gene is expressed in a liver-specific manner. Several regions homologous to known response elements were identified upstream of the C4BP gene including a strong hepatocyte nuclear factor 1 binding site and four putative glucocorticoid response elements. Furthermore, dexamethasone increased C4BP mRNA and protein levels in the mouse liver cell line, NMuLi. The stimulation of C4BP gene expression was rapid and independent of protein synthesis. These results suggest dexamethasone induction of the C4BP gene is a primary response and therefore a transcriptional effect. Inhibition of the dexamethasone effect on C4BP by actinomycin D supports this theory. These studies also provide evidence that, for optimal induction of the C4BP gene, the glucocorticoid receptor complex may cooperatively interact with accessory transcription factors. It is likely that stimulation of C4BP gene expression by dexamethasone may allude to a mechanism by which glucocorticoids exert their anti-inflammatory effects.

Animals↗

PubMed1991

Structural features of the human C3 gene: intron/exon organization, transcriptional start site, and promoter region sequence.

The third component of human complement (C3) is a key molecule in the activation of the complement cascade. C3 cDNA fragments were used to identify seven cosmid clones that covered all but 1 kilobase pair (kb) of the C3 gene. The remainder of the gene was cloned by using the polymerase chain reaction. These clones were used to identify the intron/exon boundaries and to map the gene. The C3 gene is 42 kb in length and comprises 41 exons ranging in size from 52 to 213 base pairs (bp). The transcription start site was identified by primer extension, and approximately 1 kb of DNA upstream of this site was sequenced. Putative TATA and CAAT boxes were identified along with a number of regions that shared homology with known regulatory sequences. These include responsive elements for interferon-gamma, interleukin-6, nuclear factor kappa B, estrogen, glucocorticoids and thyroid hormone. Several of these agents have been shown to affect C3 synthesis and mRNA levels. The sizes of the exons in C3 were compared to those of C4 and alpha 2-macroglobulin (alpha 2M). Thirty-nine of 41 exons in C4 were found to be of similar size to the analogous ones in C3, and two-thirds of those in alpha 2M were also similarly sized, supporting the hypothesis that these genes arose from a common ancestor.

Amino Acid Sequence↗

PubMed1989

Complement biosynthesis in human synovial tissue.

Molecular biological and immunochemical techniques have been used to study the synthesis of complement components by synovial tissue from patients with rheumatoid arthritis or osteoarthritis and by normal synovial tissue from a patient undergoing patellectomy. Using Northern and dot-blot analyses, mRNAs coding for C1-inhibitor, C2, C3, C4 and factor B have been detected, but not for C5. Quantitative analyses of the data have not shown any significant differences in the steady state levels of any of the mRNAs in synovium from rheumatoid arthritis and osteoarthritis patients. When synovial membrane fragments from rheumatoid arthritis, osteoarthritis patients or normal synovium were cultured in vitro, synthesis of C1-inhibitor, C2, C3, C4 and factor B detected by ELISA and C2, C3 and factor B were shown to be functionally active. This study thus provides conclusive evidence that synthesis of complement components occurs locally within normal and inflamed synovial tissue. The local synthesis of complement within normal synovial joints may be of importance in their defence against infection, whereas in inflamed joints it may contribute to the inflammatory response.

Arthritis, Rheumatoid↗