Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts
Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro..cap alpha..1(I) and pro..cap alpha..2(I) mRNAs but not ..beta..-actin mRNA. Fibroblasts receiving dexamethasone and (5,6-/sup 3/H)uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not ..beta..-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, the authors determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the ..cap alpha..2(I) procollagen gene. Nuclear protein blots were probed with the /sup 32/P-end-labeled pBR322 vector DNA and /sup 32/P-end-labeled ..cap alpha..2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the ..cap alpha..2(I) procollagen promoter containing DNA were calculated.
- Research Organization:
- Univ. of Vermont, Burlington (USA)
- OSTI ID:
- 7253561
- Journal Information:
- Biochemistry; (United States), Vol. 27:8
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DEXAMETHASONE
BIOLOGICAL EFFECTS
PROTEINS
GENE REGULATION
MOLECULAR WEIGHT
CELL NUCLEI
CHICKENS
COLLAGEN
DNA
FIBROBLASTS
GLUCOCORTICOIDS
IN VITRO
LABELLED COMPOUNDS
PHOSPHORUS 32
SKIN
TRANSCRIPTION
TRITIUM COMPOUNDS
URIDINE
ADRENAL HORMONES
ANIMAL CELLS
ANIMALS
AZINES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIRDS
BODY
CELL CONSTITUENTS
CONNECTIVE TISSUE CELLS
CORTICOSTEROIDS
DAYS LIVING RADIOISOTOPES
FOWL
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPES
KETONES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PHOSPHORUS ISOTOPES
PREGNANES
PYRIMIDINES
RADIOISOTOPES
RIBOSIDES
SCLEROPROTEINS
SOMATIC CELLS
STEROIDS
URACILS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques