Procollagen Alpha Research Articles

Proteomics analysis of differentially expressed metastasis-associated proteins in adenoid cystic carcinoma cell lines of human salivary gland

Metastasis is the most insidious and life threatening aspect of cancers. However little is known about the molecular mechanisms of tumor metastasis. A poorly metastatic Acc-2 cell line and highly metastatic Acc-M cell line were selected as an experimental model to study on metastatic mechanisms and antimetastatic procedures. In the present study, two-dimensional gel electrophoresis and mass spectrometry are combined to approach the protein profiles associated with tumor metastasis between Acc-2 and Acc-M cell lines. Image analysis of silver stained 2-dimensional gels revealed that 12 protein spots showed significantly quantitative and qualitative variations and mass spectrometry is utilized to further identify these spots. Of the identified proteins, transketolase, Dim1p, v-Ha-ras oncogene, type I collagen pro alpha, tumor necrosis factor (ligand) superfamily member 4, and pirin etc, have shown associations with distinct aspect of tumor metastasis to some extent. The dissimilar expression patterns of these 12 spots indicate the different roles they may play involved in tumor metastasis.

NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II-induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen alpha1(I) mRNA expression, and secretion of TGF-beta1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox-/- mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox-/- mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle alpha-actin and expression of TGF-beta1 were reduced in p47phox-/- mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

Low tidal volume ventilation induces proinflammatory and profibrogenic response in lungs of rats.

We examined whether mechanical ventilation with low tidal volume induces polymorphonuclear infiltration and proinflammatory and profibrogenic responses in rat lungs compared dependent and nondependent lung region to expression of interleukin-1beta (IL-1beta) and alpha-1 procollagen III (PC III) mRNA. An experimental, randomized and controlled protocol with previously normal rats. Three groups of ten animals were studied. Two groups were ventilated (FIO2=0.3) in supine position for 1 h without positive end expiratory pressure, one group with a low tidal volume (6 ml/kg), and the other with a high tidal volume (24 ml/kg). In the third group animals were kept in spontaneous ventilation for 1 h. After ventilation the right lung was used to quantify polymorphonuclear infiltration. The left lung was divided into dependent and nondependent regions, and expression of IL-1beta and PC III mRNA was quantified by northern blot analysis. The group ventilated with low tidal volume had greater polymorphonuclear infiltration IL-1beta and PC III mRNA expression than the nonventilated group. Similar results were observed with high tidal volumes. There was no difference between low and high tidal volume ventilation. Expression levels of IL-1beta and PC III mRNA were higher in the nondependent region of ventilated groups and equal in the nonventilated group. Even a low tidal volume mode of mechanical ventilation induces proinflammatory and profibrogenic response, with a nondependent predominance for IL-1beta and PC III mRNA expression in supine, ventilated, previously normal rats.

Identification and Characterization of the Hepatic Stellate Cell Transferrin Receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [(3)H]Thymidine incorporation, and real-time RT-PCR for procollagen alpha1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

Disruption of tumor necrosis factor receptor p55 impairs collagen turnover in experimentally induced sclerodermic skin fibroblasts.

To determine the role of tumor necrosis factor receptor p55 (TNFRp55)-mediated signaling in the pathogenesis of scleroderma. A murine model of scleroderma that closely resembles systemic sclerosis in humans was used. Wild-type and TNFRp55-deficient (TNFRp55(-/-)) mice received a subcutaneous injection of bleomycin each day. The extent of skin fibrosis was determined by measurements of the dermal thickness, as well as histologic examinations. Expression levels of fibrogenic cytokines, procollagen alpha1, and matrix metalloproteinase 1 (MMP-1), MMP-2, and MMP-9 messenger RNA (mRNA) were analyzed, both in vivo and in vitro, by reverse transcriptase-polymerase chain reaction assay or Western blotting. TNFRp55(-/-) mice began to develop severe sclerotic changes of the dermis on day 3 of the subcutaneous injections of bleomycin, while wild-type mice did not. The expression levels of fibrogenic cytokines, procollagen alpha1, and MMP-2 and MMP-9 mRNA were unaffected in the skin of both wild-type and TNFRp55(-/-) mice, with or without bleomycin treatment. Induction of MMP-1 expression was significantly inhibited in the skin from bleomycin-treated TNFRp55(-/-) mice, and this phenomenon was also observed in vitro. These results indicated that signaling mediated by TNFRp55 plays an essential role in MMP-1 expression and a key role in the collagen degradation process in this murine model. This study might provide a basis for understanding the pathogenesis of scleroderma and formulating therapeutic intervention.

Identification, characterization and expression analysis of a new fibrillar collagen gene, COL27A1.

The fibrillar collagens provide structural scaffolding and strength to the extracellular matrices of connective tissues. We identified a partial sequence of a new fibrillar collagen gene in the NCBI databases and completed the sequence with bioinformatic approaches and 5' RACE. This gene, designated COL27A1, is approximately 156 kbp long and has 61 exons located on chromosome 9q32-33. The homologous mouse gene is located on chromosome 4. The gene encodes amino- and carboxyl-terminal propeptides similar to those in the 'minor' fibrillar collagens. The triple-helical domain is, however, shorter and contains 994 amino acids with two imperfections of the Gly-Xaa-Yaa repeat pattern. There were three sites of alternative RNA splicing, only one of which led to the intact mRNA that encodes this full-length collagen proalpha chain. Phylogenetic analyses indicated that COL27A1 forms a clade with COL24A1 that is distinct from the two known lineages of fibrillar collagens. Expression analyses of the mouse col27a1 gene demonstrated high expression in cartilage, the eye and ear, but also in lung and colon. It is likely that the major protein product of COL27A1, proalpha1(XXVII), is a component of the extracellular matrices of cartilage and these other tissues. Study of this collagen should yield insights into normal chondrogenesis, and provide clues to the pathogenesis of some chondrodysplasias and disorders of other tissues in which this gene is expressed.

The Role of Hepatic Stellate Cells and Transforming Growth Factor-β 1 in Cystic Fibrosis Liver Disease

Liver disease causes significant morbidity and mortality from multilobular cirrhosis in patients with cystic fibrosis. Abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cystic fibrosis-associated liver disease (CFLD), yet the mediators linking biliary events to fibrosis remain unknown. Activated hepatic stellate cells (HSCs) are the pre-eminent mediators of fibrosis in a range of hepatic disorders. The dominant stimulus for matrix production by HSCs is the cytokine transforming growth factor (TGF)-beta(1). In CFLD, the role of HSCs and the source of TGF-beta(1) have not been evaluated. Liver biopsy tissue obtained from 38 children with CFLD was analyzed. Activated HSCs, identified by co-localization of procollagen alpha(1)(I) mRNA and alpha-smooth muscle actin, were demonstrated as the cellular source of excess collagen production in the fibrosis surrounding the bile ducts and the advancing edge of scar tissue. TGF-beta protein and TGF-beta(1) mRNA expression were shown to be predominantly expressed by bile duct epithelial cells. TGF-beta(1) expression was significantly correlated with both hepatic fibrosis and the percentage of portal tracts showing histological abnormalities associated with CFLD. This study demonstrates a definitive role for HSCs in fibrogenesis associated with CFLD and establishes a potential mechanism for the induction of HSC collagen gene expression through the production of TGF-beta(1) by bile duct epithelial cells.

Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis.

It is now generally accepted that chronic pancreatic injury and fibrosis may result from repeated episodes of acute pancreatic necroinflammation (the necrosis-fibrosis sequence). Recent studies suggest that pancreatic stellate cells (PSCs), when activated, may play an important role in the development of pancreatic fibrosis. Factors that may influence PSC activation during pancreatic necroinflammation include cytokines known to be important in the pathogenesis of acute pancreatitis, such as tumour necrosis factor alpha (TNF-alpha), and the interleukins 1, 6, and 10 (IL-1, IL-6, and IL-10). To determine the effects of these cytokines on PSC activation, as assessed by cell proliferation, alpha smooth muscle actin (alpha-SMA) expression, and collagen synthesis. Cultured rat PSCs were incubated with cytokines for 24 hours. Cell proliferation was assessed by measuring (3)H thymidine incorporation into cellular DNA, alpha-SMA expression by western blotting, and collagen synthesis by incorporation of (14)C proline into collagenase sensitive protein. mRNA levels for procollagen alpha(1)(1) in PSCs were determined by northern and dot blotting methods. Expression of alpha-SMA by PSCs was increased on exposure to each of the cytokines used in the study. Stellate cell proliferation was stimulated by TNF-alpha but inhibited by IL-6, while IL-1 and IL-10 had no effect on PSC proliferation. Collagen synthesis by PSCs was stimulated by TNF-alpha and IL-10, inhibited in response to IL-6, and unaltered by IL-1. Changes in collagen protein synthesis in response to TNF-alpha, IL-10, and IL-6 were not regulated at the mRNA level in the cells. This study has demonstrated that PSCs have the capacity to respond to cytokines known to be upregulated during acute pancreatitis. Persistent activation of PSCs by cytokines during acute pancreatitis may be a factor involved in the progression from acute pancreatitis to chronic pancreatic injury and fibrosis.

Concentration- and composition-dependent effects of metal ions on human MG-63 osteoblasts.

Metal debris from implants has been shown to alter the function of osteoblasts in cell cultures. Its remains unclear, however, if specific forms of released ionic metals are involved in the pathogenesis of periprosthetic osteolysis. We evaluated the relative effects of ionic forms of implant metals by treating human osteoblast-like MG-63 osteosarcoma cells with eight concentrations (0.001-10.0 mM) of Cr(+3), Mo(+5), Al(+3), Ta(+5), Co(+2), Ni(+2), Fe(+3), Cu(+2), Mn(+2), Mg(+2), Na(+2), and V(+3) chloride solutions. The results demonstrated that the metal ions differentially affected osteoblast proliferation, viability, type-I collagen gene expression, and cytokine release. The metal ions were ranked in order from least to most toxic (based on a 50% reduction in viability) as follows: Na < Cr < Mg < Mo < Al < Ta < Co < Ni < Fe < Cu < Mn < V. Metal-induced decreases in osteoblast proliferation were similar in ranking. Nontoxic concentrations of metals had no effect on procollagen alpha1[I] gene expression; only at toxic concentrations did metals produce a decrease in gene expression. The most toxic metals (V, Mn, Fe, and Ni) were also the only metals found to induce IL-6 secretion on a per cell basis (of the cytokines tested, interleukin 6 (IL-6), interleukin beta 1 (IL-1beta), transforming growth factor beta 1 (TGF-beta1), and tumor necrosis factor alpha (TNF-alpha), only IL-6 was detectable in the culture medium after 48 h for any metal at any concentration). Less toxic metals (e.g., Co and Cr) had little effect on IL-6 release, even at high concentrations. In general, metal ions reduced osteoblast function (i.e., proliferation and collagen gene expression) in proportion to the degree of toxicity. These results support the hypothesis that adverse local cellular responses (particularly necrotic responses) associated with metal debris from implanted metallic devices may be due in part to metal ions released from implants or from particulate debris.

Activation of Fibroblast Procollagen α1(I) Transcription by Mechanical Strain Is Transforming Growth Factor-β-dependent and Involves Increased Binding of CCAAT-binding Factor (CBF/NF-Y) at the Proximal Promoter

During normal developmental tissue growth and in a number of diseases of the cardiopulmonary system, adventitial and interstitial fibroblasts are subjected to increased mechanical strain. This leads to fibroblast activation and enhanced collagen synthesis, but the underlying mechanisms involved remain poorly understood. In this study, we have begun to identify and characterize mechanical strain-responsive elements in the rat procollagen alpha 1(I) (COL1A1) gene and show that the activity of COL1A1 promoter constructs, transiently transfected into cardiac fibroblasts, was increased between 2- and 4-fold by continuous cyclic mechanical strain. This was accompanied by an approximately 3-fold increase in the levels of total active transforming growth factor-beta (TGF-beta) released into the medium. Inclusion of a pan-specific TGF-beta neutralizing antibody inhibited strain-induced COL1A1 promoter activation. Deletion analysis revealed the presence of two potential strain response regions within the proximal promoter, one of which contains an inverted CCAAT-box overlapping a GC-rich element. Both mechanical strain and exogenously added TGF-beta1 enhanced the binding activity of CCAAT-binding factor, CBF/NF-Y, at this site. Moreover, this element was sufficient to confer strain-responsiveness to an otherwise unresponsive SV40 promoter. In summary, this study demonstrates that strain-induced COL1A1 promoter activation in cardiac fibroblasts is TGF-beta-dependent and involves increased binding of CCAAT-binding factor at the proximal promoter. Furthermore, these findings suggest a novel and potentially important TGF-beta response element in the rat COL1A1 gene.

Premature vertebral endplate ossification and mild disc degeneration in mice after inactivation of one allele belonging to the Col2a1 gene for Type II collagen.

Skeletal tissues of mice with an inactivated allele of the Col2a1 gene for Type II collagen ("heterozygous knockout") were studied. To determine whether a heterozygous inactivation of the Col2a1 gene has a role in the etiology of spine disorders such as disc degeneration. Mutations in the COL2A1, COL11A1, COL11A2, and COL9A2 genes have been linked to spine disorders. However, the mechanism by which genetic factors lead to disc degeneration still are largely unknown. Spine tissues were studied using radiograph analyses; conventional, quantitative, and polarized light microscopy; immunohistochemistry for the major extracellular components, and in situ hybridization for procollagens alpha1(I) and alpha1(II). Voluntary running activity also was monitored in half of the mice. As the findings showed, 1-month-old heterozygous knockout mice had shorter limb bones, skulls, and spines, as well as thicker and more irregular vertebral endplates, which calcified earlier than in the control mice. They also had a lower concentration of glycosaminoglycans in the anulus fibrosus, in the endplates, and in the vertebral bone than the controls. These features in the heterozygous knockout mice were compensated by the age of 15 months. However, the long bones and skulls of the mature heterozygous mice remained shorter than those of the controls. Gene-deficient mice used the running wheel less. However, physical exercise did not induce any marked structural changes in the skeleton. Mice with heterozygous knockout of Col2a1 show subtle early skeletal manifestations that bear some resemblance to those of human spine disorders.

Modulation of Skin Collagen Metabolism in Aged and Photoaged Human Skin In Vivo

To the best of our knowledge, no study has been conducted to date to directly compare the collagen metabolism of photoaged and naturally aged human skin. In this study, we compared collagen synthesis, matrix metalloproteinase-1 levels, and gelatinase activity of sun-exposed and sun-protected skin of both young and old subjects. Using northern blot analysis, immunohistochemical stain, and Western blot analysis, we demonstrated that the levels of procollagen type I mRNA and protein in photoaged and naturally aged human skin in vivo are significantly lower than those of young skin. Furthermore, we demonstrated, by northern blot analysis, that the procollagen alpha1(I) mRNA expression of photoaged skin is much greater than that of sun-protected skin in the same individual. In situ hybridization and immunohistochemical stain were used to show that the expression of type I procollagen mRNA and protein in the fibroblasts of photoaged skin is greater than for naturally aged skin. In addition, it was found, by Western blot analysis using protein extracted from the dermal tissues, that the level of procollagen type I protein in photoaged skin is lower than that of naturally aged skin. The level of matrix metalloproteinase-1 protein and the activity of matrix metalloproteinase-2 were higher in the dermis of photoaged skin than in naturally aged skin. Our results suggest that the natural aging process decreases collagen synthesis and increases the expression of matrix metalloproteinases, whereas photoaging results in an increase of collagen synthesis and greater matrix metalloproteinase expression in human skin in vivo. Thus, the balance between collagen synthesis and degradation leading to collagen deficiency is different in photoaged and naturally aged skin.

PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction

Progressive renal disease is characterized by the induction of plasminogen activator inhibitor-1 (PAI-1), suggesting that impaired activity of the renal plasmin cascade may play a role in renal fibrosis. To test this hypothesis, the severity of renal fibrosis caused by unilateral ureteral obstruction (UUO) was compared in PAI-1 wild-type (+/+) and PAI-1 deficient (-/-) mice. The extent of interstitial inflammation and fibrosis, renal plasminogen activator and plasmin activity, and renal expression of profibrotic genes was evaluated after 3, 7, and 14 days of UUO. Renal PAI-1 mRNA levels increased 8- to 16-fold in the +/+ mice after UUO surgery, and PAI-1 protein was detected in kidney homogenates. Interstitial fibrosis was significantly attenuated in -/- mice compared with +/+ mice at day 7 and day 14, based on the interstitial area stained with picrosirius red and total kidney collagen content. However, neither the mean renal plasminogen activator nor plasmin activities were increased in -/- mice compared with +/+ mice. The number of interstitial macrophages were significantly lower in the -/- mice three and seven days after UUO; interstitial myofibroblasts were significantly fewer at three days. At the same time points, this altered interstitial cellularity was associated with a significant reduction in renal mRNA levels for transforming growth factor-beta and procollagens alpha 1(I) and alpha 1(III). These studies establish an important fibrogenic role for PAI-1 in the renal fibrogenic response. The results demonstrate that one important fibrosis-promoting function of PAI-1 is its role in the recruitment of fibrosis-inducing cells, including myofibroblasts and macrophages.

Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3.

Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non-alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade. Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic stellate cell activation (alpha-smooth muscle actin and TGF-beta expression) and collagen type I synthesis (procollagen alpha1 (I) mRNA). Lipid peroxidation occurred in and adjacent to fat-laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). alpha-Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them. These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes.

Role of myofibroblasts in tumour encapsulation of hepatocellular carcinoma in haemochromatosis.

Hepatocellular carcinoma is a common malignancy and a major complication of untreated haemochromatosis. Encapsulation of liver tumours has been associated with a better prognosis and longer disease-free periods following resection. This study investigated the source of the tumour capsule in patients with haemochromatosis and coexisting hepatocellular carcinoma and examined potential factors influencing development. Five haemochromatosis patients with encapsulated hepatocellular carcinoma were studied. Myofibroblasts were identified using combined immunohistochemistry and in situ hybridisation for alpha-smooth muscle actin and procollagen alpha1(I) mRNA, respectively. Immunohistochemistry was also performed for transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-beta receptor and malondialdehyde. Procollagen alpha1(I) mRNA co-localised to alpha-smooth muscle actin positive myofibroblasts. The number of myofibroblasts was maximal within the capsule and decreased away from the tumour. TGF-beta1 protein was expressed in iron-loaded cells in non-tumour liver at the interface of tumour capsule. PDGF-beta receptor expression was observed in mesenchymal cells in the tumour capsule and in portal tracts. Malondialdehyde adducts were observed in the tumour, non-tumour tissue and in the capsule. This study provides evidence that myofibroblasts are the cell type responsible for collagen production within the tumour capsule surrounding hepatocellular carcinoma in haemochromatosis. The production of TGF-beta1 by iron-loaded hepatic cells at the tumour capsule interface may perpetuate the myofibroblastic phenotype, resulting in the formation of the tumour capsule.

Down-regulation of procollagen alpha1[I]] messenger RNA by titanium particles correlates with nuclear factor kappaB (NF-kappaB) activation and increased rel A and NF-kappaB1 binding to the collagen promoter.

Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor kappaB (NF-kappaB), and causes an approximately 50% decrease in the steady-state messenger RNA (mRNA) level of procollagen alpha1[I]. In this study, we identify three NF-kappaB binding sites within the human procollagen alpha1[I] gene promoter, show that titanium particles stimulate their binding of the NF-kappaB subunits Rel A (p65) and NF-kappaB1 (p50), and find NF-kappaB activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF-kappaB binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF-kappaB activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF-kappaB, suggesting the involvement of redox signals in NF-kappaB-mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen alpha1[I] mRNA expression and effectively blocked the titanium-induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen alpha1[I] mRNA. Collectively, these results show that titanium particles can activate NF-kappaB signaling in osteoblasts and suggest that NF-kappaB binding to the collagen gene promoter has a functional role in the down-regulation of procollagen alpha1[I] gene transcription.

Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts.

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that oxidative stress can regulate extracellular matrix in cardiac fibroblasts. Neonatal and adult rat cardiac fibroblasts in vitro were exposed to H(2)O(2) (0.05-5 microM) or the superoxide-generating system xanthine (500 microM) plus xanthine oxidase (0.001-0.1 mU/ml) (XXO) for 24 h. In-gel zymography demonstrated that H(2)O(2) and XXO each increased gelatinase activity corresponding to matrix metalloproteinases (MMP) MMP-13, MMP-2, and MMP-9. H(2)O(2) and XXO decreased collagen synthesis (collagenase-sensitive [(3)H]proline incorporation) without affecting total protein synthesis ([(3)H]leucine incorporation). H(2)O(2) and XXO decreased the expression of procollagen alpha(1)(I), alpha(2)(I), and alpha(1)(III) mRNA but increased the expression of fibronectin mRNA, suggesting a selective transcriptional effect on collagen synthesis. H(2)O(2), but not XXO, also decreased the expression of nonfibrillar procollagen alpha(1)(IV) and alpha(2)(IV) mRNA. To determine the role of endogenous antioxidant systems, cells were treated with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DDC, 100 microM) to increase intracellular superoxide or with the glucose-6-phosphate dehydrogenase inhibitor dehydroisoandrosterone 3-acetate (DHEA; 10 microM) to increase intracellular H(2)O(2). DDC and DHEA decreased collagen synthesis and increased MMP activity, and both effects were inhibited by an SOD/catalase mimetic. Thus increased oxidative stress activates MMPs and decreases fibrillar collagen synthesis in cardiac fibroblasts. Oxidative stress may play a role in the pathogenesis of myocardial remodeling by regulating the quantity and quality of extracellular matrix.

Morphology of liver repair following cholestatic liver injury: resolution of ductal hyperplasia, matrix deposition and regression of myofibroblasts.

Myofibroblasts are the primary cells responsible for increased matrix deposition in hepatic fibrosis. Activation of hepatic stellate cells and portal fibroblasts to myofibroblasts during cholestatic liver injury is accompanied by increased expression of the activation marker, alpha-smooth muscle actin (SMA), and collagen genes. In contrast to our understanding of injury, the cellular mechanisms of liver repair are not well defined. This study was designed to examine the morphological relationship between bile duct hyperplasia, matrix deposition and myofibroblast phenotype in a model of chronic cholestatic liver injury and repair. Reversible extrahepatic obstruction was accomplished in rats using a soft vessel loop suspended from the anterior abdominal wall: duct manipulation alone was performed in sham-operated controls. After 7 days, rats were either sacrificed or decompressed by release of the loop and subsequently sacrificed 2-10 days after reversal. Liver sections were obtained for in situ hybridization for procollagen alpha1(I) mRNA, immunohistochemical staining for SMA and cytokeratin 19, and histochemical staining for reticulin. Cholestatic livers demonstrated bile duct hyperplasia, which reversed to normal within 10 days after decompression. Fibrosis was also substantially reduced during this period. SMA-positive myofibroblasts were abundant and localized to regions adjacent to proliferating ducts and excess matrix in the obstructed animals. Decompressed livers showed a dramatic time-dependent reduction in the number of SMA-positive cells and in the expression of procollagen I mRNA. Our results show that the disappearance of bile duct hyperplasia after biliary decompression is accompanied by a similarly rapid loss of SMA-positive myofibroblasts. Both cellular events may abrogate enhanced matrix synthesis and allow repair to occur.

Blocking TGF-β signaling down-regulates TGF-β1 autocrine production and collagen gene expression in keloid fibroblasts

Results: Gene expression levels of TGF-b1, TGF-b RI and TGF-b RII were enhanced (by 47%, 45% and 24% respectively vs control) at 3h strain and returned to control levels by 6h. Alpha-1 procollagen mRNA levels were elevated at both 3h and 6h (by 125% and 57% respectively vs control) with the peak at 3h. The transcription of collagen receptor DDR1 was enhanced at 3h, peaked at 6h, and remained elevated at 24h (36%, 60%, and 37% respectively vs control). Interestingly, the gene expression of the VEGF was unchanged with cellular strain.

Particulate wear debris activates protein tyrosine kinases and nuclear factor kappaB, which down-regulates type I collagen synthesis in human osteoblasts.

Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.