Glucocorticoids decrease the synthesis of type I procollagen mRNAs

Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.     

Glucocorticoid and retinoid regulation of alpha-2 type I procollagen promoter activity.

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro alpha 2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha 2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-binding activity of tightly-bound nonhistone chromosomal proteins in chicken liver chromatin.

We have isolated a nonhistone chromosomal protein fraction from chicken liver chromatin which possesses high affinity and preferential sequence DNA binding. Residually DNA-bound nonhistone chromosomal proteins after 2.0 M NaCl extraction of bulk chromatin are isolated. Bound proteins are released by dissociation of the complexes in 5.0 M urea/3.0 M NaCl. We have investigated the in vitro DNA-binding properties of this class. In contrast to other DNA-binding NHCP whose activities have been studied, direct DNA-binding activity is observed which is not abolished under conditions of high ionic strength (to 3.0 M NaCl). Strong preference in binding fractionated homologous DNA is observed, while binding of heterologous (E. Coli) DNA is negligible. The fractionation of homologous DNA permits the isolation of DNA for which this protein class displays strong binding preference, presumably through a concentration of binding sites. The composite data suggest sequence-specific interaction between this protein class and DNA, which is not abolished by high ionic strength.     

Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs. Reversal of the bleomycin effect by dexamethasone

Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.     

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(5)-5.2 X 10(6) copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; (f) actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin- bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.     

DNA-binding proteins from Novikoff hepatoma cells.

In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA. 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.     

Uncoupled expression of mRNAs for alpha 1(I) and alpha 2(I) procollagen chains in chemically transformed Syrian hamster fibroblasts

Syrian hamster embryo fibroblasts transformed by 4-nitroquinoline-1-oxide (NQT-SHE cells) failed to synthesize the pro-alpha 1(I) subunit of type I procollagen but continued to synthesize altered forms of the other subunit, pro-alpha 2(I) (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). This was unusual, since synthesis of the two subunits generally is coordinately regulated. Present experiments using cell-free translation and hybridization of RNA from normal and transformed Syrian hamster fibroblasts with labeled pro-alpha 1(I) DNA probes show that mRNA for pro-alpha 1(I) is absent from the transformant. In contrast, dot-blot and Southern blot hybridizations of cellular DNAs with pro-alpha 1(I) DNA probes demonstrated that the transformed cells contained pro-alpha 1(I) gene sequences and that the gross structure of the gene was unchanged by transformation. mRNA for the other type I procollagen subunit, pro-alpha 2(I), was present in transformed cells and the major collagenous polypeptide translated from this RNA migrated like the normal pro-alpha 2 subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translated procollagen chain was cleaved to an alpha 2(I)-sized collagen chain by pepsin at 4 degrees C. These studies provide a molecular basis for the observed collagen phenotype of NQT-SHE cells.     

Variations in ADP-ribosylation of nuclear scaffold proteins during the HeLa cell cycle

Cell cycle variations in ADP-ribosylation of nuclear scaffold proteins were determined. Nuclei of synchronized cells were isolated and labeled with [32P]NAD before nuclear scaffolds were obtained by digestion of DNA with DNase I and extraction of proteins with 2M NaCl. Autoradiograms revealed the three groups of "lamins" and a species identified as poly (ADP-ribose) polymerase to be the primary ADP-ribosylated proteins. The patterns of modification of nuclear scaffold proteins displayed similar features through the cell cycle. Radioactivity in the lamins increased from 20% in early-S phase to 40% in G1 phase of the next cell cycle.     

Evidence for pretranslational regulation of collagen synthesis by procollagen propeptides

We present, here, evidence for a pretranslational role of procollagen propeptides in the regulation of collagen synthesis. Amino- and carboxyl-terminal type I procollagen propeptides were isolated and purified from chick calvaria and tendon cultures. Human lung fibroblasts (IMR-90) were incubated in medium containing varying concentrations of propeptides. Amino-propeptides at 10 nM caused an 80% decrease in collagen synthesis compared to control. Higher concentrations of amino-propeptides did not decrease collagen synthesis further and no significant effect on non-collagen synthesis was found throughout the entire concentration range. Carboxyl-propeptides also inhibited collagen synthesis. At 10 nM, collagen synthesis was decreased by 30% and a concentration of 40 nM caused an 80% reduction. However, at the latter concentration non-collagen synthesis was also affected, decreasing by 20% relative to control. To assess possible pretranslational effects of propeptides, IMR-90 fibroblasts were treated with varying concentrations of each propeptide and levels of type I procollagen mRNA was determined by dot hybridization with a 32P-alpha 2(I) cDNA probe. Both propeptides caused significant concentration-dependent decreases in procollagen type I mRNA levels. At 10 nM, the amino-propeptide resulted in a 55% decrease in collagen mRNA levels while at 40 nM these levels decreased by 72% compared to control. Carboxyl-propeptides were also inhibitory, decreasing mRNA levels by 33% at 10 nM and 73% at 40 nM. Messenger RNA levels of a representative noncollagenous protein, beta-actin, were unaffected by either propeptide throughout the concentration range.     

Parallel regulation of procollagen I and colligin, a collagen-binding protein and a member of the serine protease inhibitor family

A potential regulatory linkage between the biosynthesis of colligin, a collagen-binding protein of the ER, and procollagen I was examined under a variety of experimental conditions. Cell lines which did not produce a significant amount of procollagen I mRNA also lacked the capacity to produce colligin mRNA. Anchorage-dependent cell lines like L6 myoblasts and normal rat kidney fibroblasts produced both colligin and procollagen I mRNA, but the level of both was concurrently reduced considerably in their ras-transformed counterparts. Similarly, during the differentiation of L6 myoblasts, levels of both colligin and procollagen declined together. Treatment of myoblasts by dexamethasone or EGF led to a decrease in the steady-state levels of procollagen I mRNA, and this was, again, accompanied by a decrease in colligin mRNA synthesis. On the other hand, when the rate of procollagen I synthesis was stimulated by treatment of myoblasts with TGF beta, it led to the concurrent augmentation of both the mRNA and protein levels of colligin. A linkage between the regulation of synthesis of procollagen I and colligin thus seems to exist. The only exception to this generalization is provided by the heat induction behavior of the two proteins. Treatment of myoblasts for a very short period leads to an increase in the synthesis of both the mRNA and protein levels of colligin. This, however, is not accompanied by a change in the mRNA levels of procollagen I. These studies establish that colligin and procollagen are generally tightly co-regulated except after heat shock, suggesting an important functional linkage.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.