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.     

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

Transfer of proalpha2(I) cDNA into cells of a murine model of human Osteogenesis Imperfecta restores synthesis of type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in vitro and in vivo

The oim mouse is a model of human Osteogenesis Imperfecta (OI) that has deficient synthesis of proalpha2(I) chains. Cells isolated from oim mice synthesize alpha1(I) collagen homotrimers that accumulate in tissues. To explore the feasibility of gene therapy for OI, a murine proalpha2(I) cDNA was inserted into an adenovirus vector and transferred into bone marrow stromal cells isolated from oim mice femurs. The murine cDNA under the control of the cytomegalovirus early promoter was expressed by the transduced cells. Analysis of the collagens synthesized by the transduced cells demonstrated that the cells synthesized stable type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in the correct ratio of 2:1. The collagen was efficiently secreted and also the cells retained the osteogenic potential as indicated by the expression of alkaline phosphatase activity when the transduced cells were treated with recombinant human bone morphogenetic protein 2. Injection of the virus carrying the murine proalpha2(I) cDNA into oim skin demonstrated synthesis of type I collagen comprised of alpha1 and alpha2 chains at the injection site. These preliminary data demonstrate that collagen genes can be transferred into bone marrow stromal cells as well as fibroblasts in vivo and that the genes are efficiently expressed. These data encourage further studies in gene replacement for some forms of OI and use of bone marrow stromal cells as vehicles to deliver therapeutic genes to bone.     

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.     

A heterozygous collagen defect in a variant of the Ehlers-Danlos syndrome type VII. Evidence for a deleted amino-telopeptide domain in the pro-alpha 2(I) chain

A structural defect in the alpha 2(I) chain of type I collagen was characterized in a new case of the Ehlers-Danlos syndrome type VII. The patient's skin, fascia, and bone collagens all showed an abnormal additional chain, pN-alpha 2(I)s, running slower than the alpha 2(I) chain on electrophoresis. The extension was shown to be on the amino-terminal fragment of pN-alpha (I)s by cleavage with human collagenase, but pepsin was unable to convert pN-alpha 2(I)s to alpha 2(I). Skin collagen was 4-fold more extractable and contained fewer beta-dimers and a lower concentration of cross-linking amino acids than control skin collagen. Electron micrographs of both dermis and bone showed markedly irregular ragged outlines of the collagen fibrils in cross-section, although the patient had no clinical signs of bone disease. Procollagen secreted by her skin fibroblasts in culture showed equal amounts of the normal and abnormal alpha 2(I) chains on pepsin digestion. Before pepsin, the pN-alpha 2(I) component ran as a doublet on electrophoresis; pepsin removed only the normal slower chain. The suspected deletion in pN-alpha 2(I)s was traced by CNBr peptide analysis to the N-propeptide fragment, which behaved on electrophoresis about 15-20 residues smaller than that from the normal pN-alpha 2(I) chain. The simplest genetic explanation is a spontaneous heterozygote in which one normal and one abnormal allele for the pro-alpha 2(I) gene are expressed, the protein defect being a deletion of the junction domain that spans the N-propeptidase cleavage site and the N-telopeptide cross-linking sequence.     

Effects of procollagen peptides on the translation of type II collagen messenger ribonucleic acid and on collagen biosynthesis in chondrocytes

Type II procollagen messenger ribonucleic acid (mRNA) was isolated from chick sternum and rat chondrosarcoma cells and translated in a reticulocyte lysate cell-free system. A high molecular weight band was identified as type II procollagen by gel electrophoresis, collagenase digestion, and specific immunoprecipitation. The translation of type II mRNA was specifically inhibited by addition of type I procollagen amino-terminal extension peptide. When this peptide was added to the media of cultured fetal calf chondrocytes, chick sternal chondrocytes, or chick tendon fibroblasts, no inhibition of collagen synthesis was evident. These data suggest a general regulation of collagen biosynthesis by these peptides in the cell-free translation system. However, as indicated by the cell culture experiments, cellular characteristics and evolutionary divergence of animal species seem to restrict the effect of the peptides.     

Activation of type I collagen genes in cultured scleroderma fibroblasts

Fibroblasts cultured from affected skin areas of five patients with cutaneous scleroderma were found to produce increased amounts of collagen when compared with nonaffected control cells. Total RNA was isolated from the cultures and analyzed for its level of pro alpha 1 (I)collagen mRNA by hybridization of RNA blots with a cloned cDNA probe. The levels of pro alpha 1 (I)collagen mRNAs relative to total RNA were two- to sixfold higher in the samples from affected cells, accounting for the increased synthesis of type I collagen. Cytoplasmic dot hybridizations were performed to measure the cellular content of pro alpha 1 (I)collagen mRNA: up to ninefold increases in the level of this mRNA per cell were found. Upon subculturing, scleroderma fibroblasts were found to reduce gradually the increased synthesis of collagen to the level of nonaffected controls by the tenth passage. The levels of type I collagen mRNAs were also reduced, but more slowly. The results suggest that in scleroderma fibroblasts the genes for type I collagen are activated at procollagen mRNA level or that they are more stable and that the activating factors are lost during prolonged cell culture because cells from affected areas lose their activated state.     

Differential expression of dentin matrix protein 1, type I collagen and osteocalcin genes in rat developing mandibular bone

The expression of dentin matrix protein 1 (Dmp1) mRNA has been compared with that of type I collagen and osteocalcin mRNAs during bone formation in the rat mandible, using in situ hybridization. At embryonic day 15 (E15), type I collagen and osteocalcin mRNAs were expressed by the majority of newly-differentiated osteoblasts attached to unmineralized bone matrices, whereas Dmp1 mRNA expression was confined to only a few osteoblasts. Expression of these genes increased as the number of osteoblasts increased in specimens from E16 to E18. At E20, expression of Dmp1, type I collagen and osteocalcin was also observed in osteocytes. Dmp1 expression continued in osteocytes as they matured up to the 90-day-old specimens, whereas type I collagen and osteocalcin expression in osteocytes almost disappeared at 30 days of postnatal life. In contrast, osteoblasts continued to express type I collagen and osteocalcin in 90-day-old rats, but transiently expressed Dmp1 mRNA, which was seen in the minority of osteoblasts at 14 days of postnatal life. These data show that the developmental expression patterns of Dmp1 in osteogenic differentiation differ from those of type I collagen and osteocalcin, and Dmp1 appears to be expressed by osteocytes throughout ossification in the skeleton. These observations indicate that Dmp1 may serve unique biological functions in osteocyte and bone metabolism.     

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.     

Mechanisms of stimulation of collagen synthesis during chick embryonic skin development.

To study how collagen synthesis is regulated in developing chick embryonic skin, hydroxyproline synthesis, incorporation of proline, and translational activity and content of collagen mRNA in 12-, 15-, and 18-day skins were determined and compared with each other. Hydroxyproline synthesis in the 18-day skins was markedly increased over that in the 12-day skins, whereas proline incorporation was moderately increased. The increase in collagen synthesis from day 15 to 18 was accompanied by increases in both the translational activity and the content of type I procollagen mRNA, with a selective increase in the lower-molecular-weight species of pro alpha 1 (I) collagen mRNA. In contrast, the stimulation of collagen synthesis from day 12 to day 15 did not parallel the levels of type I procollagen mRNA. These results suggest that the stimulation of collagen synthesis is regulated by collagen mRNA levels only in the later stage of development (from day 15 to day 18). Both the collagen synthesis and type I procollagen mRNA levels in the fibroblasts isolated on each corresponding day were constant. The difference in collagen synthesis under two different culture conditions suggests that cell-matrix interaction and/or some serum factors, including several growth factors, are essential for the marked stimulation of collagen synthesis observed in 12- to 18-day skin.     

Tissues formed during distraction osteogenesis in the rabbit are determined by the distraction rate: localization of the cells that express the mRNAs and the distribution of types I and II collagens

An experimental model of leg lengthening was used to study the morphology of, the collagenous proteins present, and the collagen genes expressed in the regenerating tissue following 20% lengthening at four different distraction rates. At a distraction rate of 0.3 mm/day (8 weeks distraction), the regenerate consists of intramembranous bone and localized areas of fibrocartilage. At rates of 0.7 (4 weeks) and 1.3 mm/day (2 weeks), the bone that grows from the cut ends of the cortical bone is separated by fibrous tissue and cartilage is present. At 2.7 mm/day (1 week), only fibrous tissue and sparse bone are present. Type I collagen is present in the matrices around the cells expressing its mRNA and similarly, type II collagen is located around the chondrocytes. Type I collagen mRNA is expressed predominantly by the fibroblasts in the fibrous tissue, the bone surface cells and to a reduced extent by the osteocytes. Type II collagen mRNA is expressed by chondrocytes. The results suggest that osteoblasts and chondrocytes within the regenerate originate from the same pool of progenitor cells, and the differentiation of these cells and the expression of types I and II collagen genes are altered by different rates of distraction. These observations suggest that the optimal rate of distraction in the model is 0.7 mm/day.     

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 alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]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, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled 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 alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.