Gene expression of type I and type III collagen by mechanical stretch in anterior cruciate ligament cells

Mechanical stretch affects the healing and remodeling process of the anterior cruciate ligament (ACL) after surgery in important ways. In this study, the effects of mechanical stress on gene expression of type I and III collagen by cultured human ACL cells and roles of transforming growth factor (TGF)-beta1 in the regulation of mechanical strain-induced gene expression were investigated. Uniaxial cyclic stretch was applied on ACL cells at 10 cycles/min with 10% length stretch for 24 h. mRNA expression of the type I and type III collagen was increased by the cyclic stretch. TGF-beta1 protein in the cell culture supernatant was also increased by the stretch. In the presence of anti-TGF-beta1 antibody, stretch-induced increase in type I and type III mRNA expression was markedly ablated. The results suggest that the stretch-induced mRNA expression of the type I and type III collagen is mediated via an autocrine mechanism of TGF-beta1 released from ligament cells.     

Strain-related collagen gene expression in human osteoblast-like cells

The gene expression of cells in the musculoskeletal system, such as in bone, cartilage, ligament and tendon, is profoundly affected by mechanical loading. Previous studies have demonstrated that the expression of many genes, including collagen types I and III, are affected by mechanical strain in diverse cell types, such as human osteoblast-like SaOs-2 cells. However, whether the effect of mechanical loading on collagen gene expression is strain-related remains unclear. The goal of this study was to determine the relationship between mechanical strain and the gene expression of collagen types I and III in SaOs-2 cells. A Flexercell cellular mechanical loading system was used to subject SaOs-2 cells to equibiaxial cyclic tensile stress at a rate of 0.5 Hz with various strains of 5%, 7.5%, 10%, and 12.5% for 24 h. The relative amount of mRNA of both collagen I and collagen III increased at 5% strain compared with that of the control. As the strain increased, the relative amount of mRNA of collagen I remained stable at strain levels up to 12.5%. However, the mRNA for collagen III began to drop when the strain was greater than 5%, until a 10% strain was reached. From the application of a 10% strain through the maximum loading of a 12.5% strain, the relative amount of collagen III mRNA remained stable at amounts lower than that of the control. Thus, the gene expression of collagen types I and III responds differentially to mechanical strain at various magnitudes.     

Similar, but not identical, modulation of expression of extracellular matrix components during in vitro and in vivo aging of human skin fibroblasts.

Regulation of the synthesis of procollagen and other extracellular matrix components was examined in human skin fibroblasts obtained from donors of various ages, from fetal to 80 years old (in vivo aged), and in fetal fibroblasts at varying passage levels (in vitro aged). Growth rates and saturation densities of fibroblasts decreased with increasing age of the donor and after passage 20 of fetal fibroblasts. The rates of collagen and proteoglycan synthesis also decreased during both types of aging to about 10-25% of the rate in early passage fetal fibroblasts, whereas the synthesis of total noncollagenous proteins was not greatly affected. Decreased collagen synthesis in both types of aging was correlated with lower steady-state levels of mRNAs for the two subunits of type I procollagen mRNA, although their regulation was not coordinate. Type III collagen mRNA levels also declined in both types of aging. The concentration of fibronectin mRNA also decreased during in vitro aging but more rapidly than the collagen mRNAs, whereas in fibroblasts from 51-80-year-old donors, it was similar to or higher than in early passage fetal fibroblasts. This study suggests that the decreased synthesis of procollagen and proteoglycans in in vivo aged fibroblasts represents changes that are responsible for intrinsic degenerative changes that occur in human skin during aging. Furthermore, although in vitro and in vivo aging were similar in many respects, they were not equivalent, as evidenced by the differences in regulation of fibronectin expression.     

mRNA levels for alpha-subunit of prolyl 4-hydroxylase and fibrillar collagens in immobilized rat skeletal muscle.

There is evidence that immobilization causes a decrease in total collagen synthesis in skeletal muscle within a few days. In this study, early immobilization effects on the expression of prolyl 4-hydroxylase (PH) and the main fibrillar collagens at mRNA and protein levels were investigated in rat skeletal muscle. The right hindlimb was immobilized in full plantar flexion for 1, 3, and 7 days. Steady-state mRNAs for alpha- and beta-subunits of PH and type I and III procollagen, PH activity, and collagen content were measured in gastrocnemius and plantaris muscles. Type I and III procollagen mRNAs were also measured in soleus and tibialis anterior muscles. The mRNA level for the PH alpha-subunit decreased by 49 and 55% (P < 0.01) in gastrocnemius muscle and by 41 and 39% (P < 0.05) in plantaris muscle after immobilization for 1 and 3 days, respectively. PH activity was decreased (P < 0.05-0.01) in both muscles at days 3 and 7. The mRNA levels for type I and III procollagen were decreased by 26-56% (P < 0.05-0.001) in soleus, tibialis anterior, and plantaris muscles at day 3. The present results thus suggest that pretranslational downregulation plays a key role in fibrillar collagen synthesis in the early phase of immobilization-induced muscle atrophy.     

Immune interferon inhibits collagen synthesis by rheumatoid synovial cells associated with decreased levels of the procollagen mRNAs

Recombinant immune interferon, (interferon-gamma, IFN-gamma) inhibits types I and III collagen synthesis by rheumatoid synovial fibroblast-like cells in culture. This decrease is associated with a decrease in the levels of types I and III procollagen mRNAs in these cells as measured by dot blot hybridization. In the control synovial cells the level of alpha 2(I) mRNA is disproportionately high compared with that of alpha 1(I) or alpha 1(III) mRNA, and IFN-gamma suppresses the level of alpha 1(I) and alpha 1(III) mRNA to a greater extent than that of alpha 2(I) mRNA. The lymphokine, IFN-gamma, may thus have a role in the regulation of collagen synthesis in inflammatory joint disease and other conditions.     

Changes in the distribution of fibrillar collagens in the collateral and cruciate ligaments of the rabbit knee joint during fetal and postnatal development

Summary The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Types I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years, the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.     

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.     

Regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D3 in human osteosarcoma cells

Synthesis of type I and III collagens has been examined in MG-63 human osteosarcoma cells after treatment with the steroid hormone, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Analysis of total [3H]proline-labeled proteins and pepsin-derived collagens revealed that 1,25-(OH)2D3 selectively stimulated synthesis of alpha 1I and alpha 2I components of type I collagen after 6-12 h. Consistent with previous reports (Franceschi, R. T., Linson, C. J., Peter, T. C., and Romano, P. R. (1987) J. Biol. Chem. 262, 4165-4171), parallel increases in fibronectin synthesis were also observed. Hormonal effects were maximal (2- to 2.5-fold versus controls) after 24 h and persisted for at least 48 h. In contrast, synthesis of the alpha 1III component of type III collagen was not appreciably affected by hormone treatment. Of several vitamin D metabolites (1,25-(OH)2D3, 25-dihydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) tested for activity in stimulating type I collagen synthesis, 1,25-(OH)2D3 was found to be the most active. Analysis of collagen mRNA abundance by Northern blot hybridization indicated that both types I and III procollagen mRNAs were increased 4-fold after a 24-h exposure to 1,25-(OH)2D3. Pro alpha 1I mRNA remained elevated through the 48-h time point while pro alpha 2I and pro alpha 1III mRNAs returned to control values. These results indicate that the regulation of collagen synthesis by 1,25-(OH)2D3 is complex and may involve changes in translational efficiency as well as mRNA abundance. 1,25-(OH)2D3 also caused at least a 20-fold increase in levels of the bone-specific calcium-binding protein, osteocalcin. These results are consistent with the hypothesis that 1,25-(OH)2D3 is stimulating partial differentiation to the osteoblast phenotype in MG-63 cells.     

Mechanical stretch-induced changes in cell morphology and mRNA expression of tendon/ligament-associated genes in rat bone-marrow mesenchymal stem cells

It has been demonstrated that mechanical stimulation plays a vital role in regulating the proliferation and differentiation of stem cells. However, little is known about the effects of mechanical stress on tendon/ligament development from mesenchymal stem cells (MSCs). Here, using a custom-made cell-stretching device, we studied the effects of mechanical stretching on the cell morphology and mRNA expression of several key genes modulating tendon/ligament genesis. We demonstrate that bone-marrow-derived rat MSCs (rMSCs), when subjected to cyclic uniaxial stretching, express obvious detectable mRNAs for tenascin C and scleraxis, a unique maker of tendon/ligament formation, and significantly increased levels of type I collagen and type III collagen mRNAs. The stretched cells also orient at approximately 65 degrees with respect to the stretching direction and exhibit a more fibroblast-like morphology. Collectively, these results indicate that mechanical stretching facilitates the directed differentiation of rMSCs into tendon/ligament fibroblasts, which has potential implications for the tissue engineering of bioartificial tendons and ligaments.     

Expression of type X collagen mRNA levels in embryonic chick sternum during development

Embryonic chick sternum cartilage exhibits profound spatial and temporal changes in Type X collagen biosynthesis during development. Production of this collagen is confined to the presumptive calcification region and its expression is not acquired until stage 43. To examine the mechanisms responsible for regulation of developmental changes in biosynthetic expression of Type X collagen, we determined the levels of translatable Type X procollagen mRNA employing a cell-free translation system. We found that mRNA capable of directing Type X collagen synthesis was present exclusively in cartilage destined to undergo calcification and that its levels were nearly equivalent at all stages of development. These findings suggest that expression of Type X collagen in embryonic chick sternum is determined at the translational level.     

Peroxisome proliferator-activated receptor-γ ligands attenuate brain natriuretic peptide production and affect remodeling in cardiac fibroblasts in reoxygenation after hypoxia

Cardiac fibroblasts (CFs) participate in cardiac remodeling after hypoxic cardiac damage, and remodeling is thought to be mediated by CF synthesis of brain natriuretic peptide (BNP). It is unknown whether the peroxisome proliferator-activated receptors (PPARs), which mediate cellular signaling for growth and migration, affect BNP synthesis and whether PPARs participate in regulation of extracellular matrix protein (ECM) expression for remodeling. We examined the production of BNP in cultured neonatal ventricular CFs and its signaling system on collagen synthesis and on activation of matrix metalloproteinases (MMPs) in reoxygenation after hypoxia. BNP mRNA was detected in CFs, and a specific BNP protein, BNP1-32, was secreted into the media. Abundance of collagen I and III was increased in the media at reoxygenation. mRNA and protein levels for MMP-2 and the tissue inhibitor of metalloproteinase (TIMP)-1 were enhanced in CFs at reoxygenation. These observations also were noted in CFs after incubation with angiotensin II (10 μM) for 24 h. Pretreatment with pioglitaozone (0.1–10 μM) attenuated BNP mRNA and protein abundance of collagen III, MMP-2, and TIMP-1 in CFs at reoxygenation. The secreted BNP was also decreased by pioglitaozone in the media. Furthermore, PPAR activators inhibited reoxygenation-induced activation of nuclear factor (NF)-kB. These results demonstrate that PPAR activators inhibit BNP synthesis in CFs and imply that PPAR activators may regulate ECM remodeling partially through the NF-kB-mediated pathway.     

Specific immunohistochemical localization of Type I collagen in porcine periodontal tissues using the peroxidase-labelled antibody technique

Synopsis Antibody against Type I collagen was raised in rabbits and purified by immunoadsorption on Sepharose-conjugated Types I and III collagen. The cross-reactivity of purified antibody to Type III collagen was found to be less than 0.5% by passive haemagglutination and less than 1.5% by radioimmunoassay. When paraffin sections of fixed and decalcified pig molars were incubated with purified antibody to Type I collagen, varying degrees of staining were observed in the ligament, gingiva, bone and cementum. The periodontal ligament adjacent to bone was more widely stained than that adjacent to cementum in some regions, whereas in others, no difference in staining could be discerned between the two halves of the ligament. The lamina propria of gingiva was stained, and this appeared to be most intense in the vicinity of the overlying epithelium. The fibrous component in the endosteal spaces, the dentine and the extracellular coronal elements in the pulp were generally stained. The impression obtained from the staining pattern is that Type I collagen is not restricted to particular regions of the periodontal ligament or the lamina propria of the gingiva.