Hydralazine differentially increases mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase whereas it decreases pro alpha 1(I) collagen mRNAs in human skin fibroblasts.

We have used specific oligonucleotide probes to measure the effect of hydralazine on mRNA levels of the alpha and beta subunits of prolyl 4-hydroxylase (PH), a key post-translational modifying enzyme in collagen biosynthesis. Hydralazine exerts a paradoxical effect on collagen biosynthesis in cultured fibroblasts. Cells exposed to hydralazine synthesize substantially reduced amounts of collagen, which is severely deficient in hydroxyproline. Surprisingly, however, the level of prolyl hydroxylase activity assayed in extracts of treated cells is markedly increased, suggesting overproduction of the enzyme. Hybridization analysis indicated that in untreated cells the concentration of the alpha PH subunit mRNA was about 20-25% of the beta PH subunit mRNA concentration. Hydralazine treatment increased the mRNAs for both alpha and beta subunits of PH by three- to fourfold. A differential induction of these mRNAs was observed, however. The alpha subunit mRNA was maximally increased within 24 h, whereas the beta subunit mRNA was increased more slowly, reaching a maximum at 72 h. In contrast, the 5.8 and 4.8-kb mRNAs for pro alpha 1(I) collagen were virtually eliminated by 72 h. This study demonstrates that the increased prolyl hydroxylase activity is a direct result of hydralazine-mediated increases in steady state mRNA content for the alpha and beta subunits of this enzyme. Moreover, the earlier induction of alpha PH mRNA may provide the first evidence at the mRNA level that regulation of PH activity occurs mainly through regulation of the alpha subunit of PH. In addition, the decrease in collagen synthesis by hydralazine appears to result directly from suppression of both species of mRNA for pro alpha 1(I) collagen.     

Gamma-interferon inhibits extracellular matrix synthesis and remodeling in collagen lattice cultures of normal and scleroderma skin fibroblasts.

Three-dimensional collagen lattice cultures of fibroblasts mimic the in vivo situation better than monolayer cultures. Here, skin fibroblasts from scleroderma patients and healthy controls were cultivated in collagen lattices, and the effects of recombinant human gamma-interferon (IFN-gamma) on these cultures investigated. IFN-gamma inhibited collagen lattice retraction in a dose-dependent way at concentrations ranging from 10 to 10,000 U/ml. This effect was independent of any alteration to the cell proliferation within the lattices. The inhibition was of the same order of magnitude in normal and pathological fibroblasts. The synthesis of collagen and non-collagen proteins, particularly fibronectin, was increased in scleroderma cultures. It was inhibited in both normal and scleroderma fibroblasts by IFN-gamma, with a maximal effect at the concentration 1000 U/ml, but the inhibition of protein synthesis was far more intense in scleroderma than in normal cells. In situ hybridization, Northern blot and dot blot analyses showed that mRNA coding for pro alpha 1(I) collagen was decreased in IFN-gamma-treated cells, indicating an effect at the pretranslational level. IFN-gamma also inhibited glycosaminoglycan synthesis, but in scleroderma cells only. This study shows that IFN-gamma regulates cell behavior in three-dimensional collagen matrices: (i) it decreases protein and specifically glycosaminoglycan synthesis in scleroderma fibroblasts, (ii) it modulates the interactions between cells and matrix that lead to the retraction of the lattice. Whereas collagen synthesis is largely decreased in lattice cultures like in vivo, it remains increased in the case of scleroderma compared to normal fibroblasts and may be down-regulated by IFN-gamma. Similar conclusions may be drawn for fibronectin and glycosaminoglycans. The inhibitory effect of IFN-gamma on the retraction capacity of fibroblasts and on their ability to synthesize increased amounts of extracellular matrix macromolecules may be of potential interest for therapeutic use of IFN-gamma in scleroderma patients.     

Regulatory Role of the Conserved Stem-Loop Structure at the 5′ End of Collagen α1(I) mRNA

Three fibrillar collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen alpha1(I) reporter mRNAs in hsc's and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc's and to a lesser extent in activated hsc's and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc's and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3' untranslated region (UTR) of collagen alpha1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc's. This 3' UTR binds alphaCP, a protein implicated in increasing stability of collagen alpha1(I) mRNA in activated hsc's (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201-5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen alpha1(I) mRNA is regulated by a complex interaction between the 5' stem-loop and the 3' UTR, which may optimize collagen production in activated hsc's.     

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)     

Deficiency of tenascin-X causes a decrease in the level of expression of type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX-null fibroblasts exhibit decreased cell-matrix and cell-cell adhesion. In this study, we used a differential display technique to determine the genes involved in this process. Differential display analysis of wild-type and TNX-null fibroblasts revealed that mRNA expression level of type VI collagen alpha3 is predominantly decreased in TNX-null fibroblasts. Expression levels of mRNAs of other subunits of type VI collagen, alpha2 and alpha3 chains, were also remarkably decreased in TNX-null fibroblasts. The protein level of alpha3 chain of type VI collagen was also reduced in TNX-null fibroblasts. However, the organization of type VI collagen in the extracellular matrix of TNX-null fibroblasts was similar to that of wild-type fibroblasts. Transient expression of TNX in Balb3T3 cells caused an increase in the level of mRNA of type VI collagen compared with that in vector control and increased the promoter activity of type VI collagen alpha1 subunit gene. In addition, the expression levels of type I collagen and other collagen fibril-associated molecules such as type XII and type XIV collagens, decorin, lumican and fibromodulin in wild-type and TNX-null fibroblasts were compared. It was found that the mRNA expression levels of type I collagen and collagen fibril-associated molecules other than decorin were decreased and that the expression level of decorin was increased in TNX-null fibroblasts. The results suggest the possibility that TNX mediates not only cell-cell and cell-matrix interactions but also fibrillogenesis via collagen fibril-associated molecules.     

Interleukin-1 increases collagen production and mRNA levels in cultured skin fibroblasts

In the present study we show that highly purified human interleukin-1 increases collagen production nearly 2-fold and mRNA levels of type I and III collagen over 2.5-fold in cultured normal human dermal fibroblasts. To minimize the effects of transient prostaglanding E2 production in fibroblasts treated with interleukin-1, the cell cultures were preincubated for 24 h before these measurements were made. The effects of interleukin-1 were also tested on scleroderma fibroblasts exhibiting increased collagen production. Although collagen synthesis was stimulated by interleukin-1 to some degree, the cells grown from both affected and unaffected skin areas were found to be relatively unresponsive to the effects of interleukin-1, suggesting a role for this monokine in the earlier stages of the disease process. The results also suggest that interleukin-1 has a role in stimulation of collagen synthesis under certain normal and pathological conditions in addition to stimulating fibroblast proliferation.     

Type I collagen messenger RNA levels in experimental granulation tissue and silicosis in rats

A complementary DNA (cDNA) clone was constructed for chick pro alpha 2(I) collagen mRNA. This and previously constructed cDNA clones for chick and human pro alpha 1(I) collagen mRNAs were used to measure levels of type I procollagen messenger RNAs in two experimental models: viscose cellulose sponge-induced experimental granulation tissue and silica-induced experimental lung fibrosis in rats. Both Northern RNA blot and RNA dot hybridizations were used to quantitate procollagen mRNAs during formation of granulation tissue. The period of rapid collagen synthesis was characterized by high levels of procollagen mRNAs, which were reduced when collagen production returned to a low basal level. The rate of collagen synthesis and the levels of procollagen mRNAs during the period of rapid reduction in collagen production did not, however, parallel with each other. This suggests that translational control mechanisms are important during this time in preventing overproduction of collagen. In silicotic lungs, the early stages of fibroblast activation follow a similar path but appear faster. At a later stage, however, the RNA levels increase again and permit collagen synthesis to continue at a high rate, resulting in massive collagen accumulation.     

Regulation of collagen production and collagen mRNA amounts in fibroblasts in response to culture conditions.

Collagen synthesis and mRNA amounts for the alpha 1 and alpha 2 polypeptide chains of Type I collagen were measured in embryonic-chick tendons and in tendon cells both in suspension and in primary cultures. The percentage of protein production represented by collagen in suspension-cultured cells was initially the same as in the intact tendon; however, on an hourly basis, there was actually a steady decline in collagen production by suspended cells. Collagen production in primary cultures of chick tendon fibroblasts was decreased when compared with intact tendon, even though ascorbate-supplemented primary cultures were able to maintain higher rates of collagen production than were non-supplemented cultures. The amounts of mRNA for alpha 1(I) and alpha 2(I) polypeptide chains of collagen responded in similar fashions to different culture conditions and were compared with the amounts of mRNA for beta-actin. In primary cultures the available alpha 1 and alpha 2 collagen mRNAs support proportionately higher collagen production than in the intact tendon. However, the ratio of alpha 1/alpha 2 mRNA and polypeptide-chain synthesis did not remain 2:1, but increased with the concomitant production of Type I trimers composed of three alpha 1 chains. Removal of fibroblasts from their environment in vivo appears to alter the amounts of mRNA for alpha 1 and alpha 2 chains and to alter the utilization of those mRNAs for polypeptide synthesis.     

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.     

Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.     

Existence of malfunctioning pro alpha2(I) collagen genes in a patient with a pro alpha 2(I)-chain-defective variant of Ehlers-Danlos syndrome

Collagen synthesis was examined in skin fibroblasts from a patient with a variant of Ehlers-Danlos syndrome. The relative rate of collagen synthesis to total protein synthesis in the patient's fibroblasts was always one-half of that in fibroblasts from normal controls. Total collagen synthesis, as assessed by quantification of total hydroxyproline, was also significantly lower than that of controls, indicating that the rate of collagen synthesis by the patient's fibroblasts was decreased compared with that by normal fibroblasts. Analysis of procollagen and collagen components showed the absence of the pro alpha 2(I) chain and its derivatives. Dot-blot and Northern-blot analyses showed the patient's fibroblasts to contain less than 10% of the mRNAs for pro alpha 2(I) found in control fibroblasts. In spite of these results, Southern blot analysis of genomic DNA indicated the presence of the same number of genes for the pro alpha 2(I) collagen chain in the patient's fibroblasts as in control fibroblasts, suggesting malfunctioning pro alpha 2(I) collagen genes as the cause for failure of the patient's fibroblasts to synthesize pro alpha 2(I) collagen chains.