Suppression of type I collagen production by microRNA-29b in cultured human stellate cells

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through imperfect base pairing with the 3′ untranslated region (3′UTR) of target mRNA. We studied the regulation of alpha 1 (I) collagen (Col1A1) expression by miRNAs in human stellate cells, which are involved in liver fibrogenesis. Among miR-29b, -143, and -218, whose expressions were altered in response to transforming growth factor-β1 or interferon-α stimulation, miR-29b was the most effective suppressor of type I collagen at the mRNA and protein level via its direct binding to Col1A1 3′UTR. miR-29b also had an effect on SP1 expression. These results suggested that miR-29b is involved in the regulation of type I collagen expression by interferon-α in hepatic stellate cells. It is anticipated that miR-29b will be used for the regulation of stellate cell activation and lead to antifibrotic therapy.     

Cellular autofluorescence following ionizing radiation

Cells often autofluoresce in response to UV radiation excitation and this can reflect critical aspects of cellular metabolism. Here we report that many different human and murine cell types respond to ionizing radiation with a striking rise in autofluorescence that is dependent on dose and time. There was a highly reproducible fluorescent shift at various wavelengths, which was mirrored by an equally reproducible rise in the vital intracellular metabolic co-factors FAD and NADH. It appears that mitochondria, metabolism and Ca(2+) homeostasis are important for this to occur as cells without mitochondria or cells unable to alter calcium levels did not behave in this way. We believe these radiation-induced changes are of biological importance and that autofluorescence may even provide us with a tool to monitor radiation responses in the clinic.     

Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta

Human platelet-derived transforming growth factor-beta (TGF-beta 1) increases the accumulation of the extracellular matrix proteins, fibronectin and type I collagen, in mesenchymal and epithelial cells. To determine the basis for this effect, we have examined the levels of mRNAs corresponding to fibronectin and alpha 2(I) procollagen in NRK-49 rat fibroblasts and L6E9 rat myoblasts treated with TGF-beta 1. TGF-beta 1 increased severalfold the levels of mRNAs for both proteins. The kinetics of this effect were similar for both mRNA species. The increase in fibronectin and alpha 2(I) procollagen mRNAs was detectable 2 h after addition of TGF-beta 1 to the cells and their maximal levels remained constant for several days. Actinomycin D, but not cycloheximide, inhibited the increase in fibronectin and alpha 2(I) procollagen mRNA levels induced by TGF-beta 1. The results indicate that TGF-beta 1 controls the composition and abundance of extracellular matrices at least in part by inducing a coordinate increase in the levels of fibronectin and type I collagen mRNAs.     

Human mammary cell survival following ionizing radiation

We have developed and evaluated methods of culturing defined stromal and epithelial populations of normal human breast cells. These cell populations were used to generate radiation dose/survival curves. The epithelial cell population required specific hormones, growth factors, and conditioned media, as well as fibroblast feeder layers for clonal growth. Stromal cells grew well in a less complex medium. The stromal and parenchymal cell populations of the normal human breast were characterized by light and electron microscopy, immunohistochemical human fibronectin staining, gamma glutamyltranspeptidase histochemical staining, and cell sizing. Survival curves were generated using cells from four donors. The average D0 for epithelial cells was 122 cGy, with an average n value of 2.4. The average D0 and n values for stromal cells were 114 cGy and 2.0. The survival of human breast epithelial cells is compared to that of the cells of the rat mammary gland. The D0 values of both species are essentially the same, while the n value for human epithelial cells is lower. This difference in the n value may be a species specific response to radiation, or may merely reflect a difference in the two assay systems used to generate the survival curves.     

Regulation of collagen synthesis by ascorbic acid. Ascorbic acid increases type I procollagen mRNA

Ascorbic acid specifically stimulates collagen production in cultured human skin fibroblasts, an effect that appears to be independent of its cofactor role in prolyl and lysyl hydroxylation. In order to investigate the level of regulation of ascorbic acid on collagen synthesis, we have translated mRNA in a cell-free system derived from rabbit reticulocytes. Total RNA was prepared from normal human skin fibroblasts and similar fibroblasts which had been exposed to 100 uM ascorbic acid for four days. Ascorbic acid treatment resulted in a twofold stimulation of procollagen mRNA whereas non-collagenous mRNA was unchanged. These results reveal that ascorbic acid has a preferential stimulating effect on type I procollagen mRNA.     

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.     

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both α1 and α2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-β1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-β receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of α2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.     

Differential expression of type I and type III collagen genes during tooth development

Collagen gene expression during mouse molar tooth development was studied by quantitative in situ hybridization techniques. Different expression patterns of type I and type III collagen mRNAs were observed in the various mesenchymal tissues that constitute the tooth germ. High concentration for pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were found within the osteoblasts. We found that the cellular content of type I collagen mRNAs in the odontoblasts varies throughout the tooth formation: whereas mRNA concentration for pro-alpha 1(I) collagen decreases and that of pro-alpha 2(I) increases, during postnatal development. Moreover, different amounts of pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were observed in crown and root odontoblasts, respectively. Type III collagen mRNAs were detected in most of the mesenchymal cells, codistributed with type I collagen mRNAs, except in odontoblasts and osteoblasts. Finally, this study reports differential accumulation of collagen mRNAs during mouse tooth development and points out that type I collagen gene expression is regulated by distinct mechanisms during odontoblast differentiation process. These results support the independent expression of the collagen genes under developmental tissue-specific control.     

Pancreatic cancer cells respond to type I collagen by inducing snail expression to promote membrane type 1 matrix metalloproteinase-dependent collagen invasion

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.     

Down-regulation of BRCA2 expression by collagen type I promotes prostate cancer cell proliferation

BRCA2 is a tumor suppressor gene that when mutated confers an increased susceptibility to developing breast and prostate carcinoma. Besides its role in mediating DNA repair, new evidence suggests that BRCA2 may also play a role in suppressing cancer cell growth. Because altered interactions between neoplastic cells and the surrounding extracellular matrix (ECM) play a pivotal role in unchecked cancer cell proliferation and metastatic progression, we hypothesized that the ECM may have an effect in BRCA2 expression. By using normal and prostate carcinoma cell lines, we demonstrated that although normal cells transiently increase BRCA2 protein levels when adhering to the ECM protein collagen type I (COL1), carcinoma cells exhibit a significant reduction in BRCA2 protein. This aberrant effect is independent from de novo protein synthesis and results from COL1-beta(1) integrin signaling through phosphatidylinositol (PI) 3-kinase leading to BRCA2 ubiquitination and degradation in the proteasome. BRCA2 protein depletion after cancer cell adhesion to COL1 or in small RNA interference assays triggers new DNA synthesis, a trophic effect that is abrogated by recombinant BRCA2 expression. Blocking or inhibiting beta(1) integrin, PI 3-kinase, or proteasome activity all have a negative effect on COL1-mediated DNA synthesis in cancer cells. In normal cells, the transient increase in BRCA2 expression is independent from beta(1) integrin or PI 3-kinase and has no effect in cell proliferation. In summary, these results unravel a novel mechanism whereby prostate carcinoma cell proliferation is enhanced by the down-regulation of BRCA2 expression when interacting with COL1, a major component of the ECM at osseous metastatic sites.     

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.     

Mineralization of type I collagen

It was previously found that the lateral spacing of the collagen molecules in wet mineralized tissues is exactly proportional to the inverse wet density. Several properties were investigated and the same type of relationship was observed each time. A possible explanation is offered. It is hypothesized that mineral is deposited initially in the extrafibrillar space so as to isolate the fibrils. Further deposition reduces the net free fibril volume thereby decreasing the spacing between collagen molecules. The linear relationship is derived from density considerations together with limitations on the collagen packing structure described as the generalized packing model. Three experimental situations were studied: lateral spacing wet tissue versus density; lateral spacing dry tissue versus density; and lateral spacing versus water content. The observed variations of the spacing can be attributed to a structure where the mass of the tissue remains constant but the volume decreases linearly with increasing mineral content.