Procollagen Gene Research Articles

Effects of Growth Hormone Transgene Expression on Vertebrae in a Mouse Model of Osteogenesis Imperfecta

A human growth hormone transgene was bred into mice of the Cola2oim (oim) lineage. Caudal (tail) vertebrae from male and female mice at early skeletal maturity and at midlife were evaluated for physical and biomechanical properties. To test whether constant low-level growth hormone expression within the marrow could improve structural or material properties of caudal vertebrae in oim mice. A spontaneous genetic defect in a type I procollagen gene created the oim mouse model for osteogenesis imperfecta. Bones of heterozygous oim mice are biomechanically inferior to wild-type controls. Bone marrow expression of human growth hormone was demonstrated previously to enhance bone deposition and structural biomechanical properties in caudal vertebrae of transgenic mice. Compression tests were performed individually on three caudal vertebrae (Ca4, 5, and 6) from each mouse to determine their structural biomechanical properties. Volumetric and mineral content measurements were also made. In a subset of vertebrae, the ashing measurements were confirmed and extended by peripheral quantitative tomographic scanning, which also allowed calculation of the failure stress. Heterozygous oim mouse vertebrae had structural and material properties inferior to the wild-type controls. Growth hormone transgene expression increased the size and mineral content of the vertebrae from mutant mice, and increased biomechanical structural values for maximum load and energy to failure. Failure stress was not improved. Growth hormone stimulation of size and bone mineral content of osteogenesis imperfecta mutant mouse caudal vertebrae contributed to their improved performance in axial compression. There was no evidence for improved material properties, however.

Vein wall remodeling after deep vein thrombosis involves matrix metalloproteinases and late fibrosis in a mouse model

Deep venous thrombosis (DVT) confers vein wall injury associated with fibrosis and extracellular matrix (ECM) turnover, likely mediated by matrix proteases. This study investigated the expression of proteases and collagen involved in early vein wall remodeling. In the mouse, DVT was produced by ligation of the infrarenal inferior vena cava (IVC) or sham operation, and tissue was harvested at 4, 8, and 12 days. The vein wall tissue was processed for real-time reverse transcriptase-polymerase chain reaction (6 to 8 per time point), Western immunoblotting (5 per time point), and gelatin zymography (5 per time point). Analysis of variance was used for multiple comparisons, and a P < .05 was significant. Thrombus resolution was documented by a 38% decrease in the thrombosed IVC weight from day 4 to day 12 (P = .007). Total vein wall collagen increased over time, with a corresponding increase in procollagen I and III, and expression peaked at 12 days (24-fold and 6.1-fold, respectively, P < .02). Matrix metalloproteinase-2 (MMP-2) gene expression was 23-fold greater at 12 days after thrombus formation compared with sham or 4 days after thrombosis (P < .05). Total MMP-2 activity was also significantly elevated at 12 days compared with sham (P < .05). MMP-9 expression was 19-fold and 27-fold higher at days 4 and 8, respectively, relative to sham (P < .05), with no difference in activity. MMP-14 expression was twofold to 3.6-fold greater at day 12 compared with earlier time points and shams (P < .001), but no differences in protein levels were found. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) protein levels were not significantly different from sham over time; however, the ratio of uPA to PAI-1 was decreased through 8 days. Vein wall remodeling after DVT is similar to wound healing and is associated with increased procollagen gene expression and total collagen. It is also associated with increased early MMP-9 expression, followed by MMP-2 expression and activity after DVT resolution. Deep vein thrombosis is an often neglected problem that long term is associated with the postphlebitic syndrome of limb swelling, pain, and often ulceration. The basic mechanisms of the vein wall damage that results have not been delineated. The following study describes the vein wall matrix metalloproteinase gene and activity response induced over time in the vein wall after DVT. Additionally, the corresponding collagen upregulation and proximate plasmin system mediators are determined. With this knowledge, potential therapies to reduce vein wall injury directly might be possible.

Transforming Growth Factor-β1 Drives Airway Remodeling in Cigarette Smoke–Exposed Tracheal Explants

Small airway remodeling (SAR) is an important cause of airflow obstruction in cigarette smokers, but whether SAR represents a response to smoke-evoked inflammation or is directly mediated by smoke-induced growth factor production is disputed. To examine this process, we exposed rat tracheal explants, a model free of exogenous inflammatory cells, to cigarette smoke in vitro. Cigarette smoke caused release of active transforming growth factor (TGF)-beta1, and this was prevented by the oxidant scavenger tetramethythiourea. Nuclear immunostaining for phospho-Smad2, a TGF-beta downstream signaling molecule, was present in epithelial and interstitial cells within 1 h after exposure. Smoke caused upregulation of gene expression of connective tissue growth factor (CTGF), a mediator of TGF-beta fibrogenic effects, within 2 h, and upregulation of procollagen gene expression at 24 h; both changes could be prevented by the TGF-beta antagonist fetuin (alpha2-HS-glycoprotein). In a cell-free system, recombinant human TGF-beta latency-associated peptide was oxidized by cigarette smoke, and smoke released active TGF-beta1 from recombinant latent TGF-beta1 via an oxidant mechanism. These experiments suggest that SAR in cigarette smokers may be caused by direct, smoke-mediated, oxidant-driven induction of growth factor signaling in the airway wall, and that SAR does not necessarily require exogenous inflammatory cells.

Reduced ABCA1-Mediated Cholesterol Efflux and Accelerated Atherosclerosis in Apolipoprotein E–Deficient Mice Lacking Macrophage-Derived ACAT1

Macrophage acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) and apolipoprotein E (apoE) have been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation. Deletion of either ACAT1 or apoE results in increased atherosclerosis in hyperlipidemic mice, possibly as a consequence of altered cholesterol processing. We have studied the effect of macrophage ACAT1 deletion on atherogenesis in apoE-deficient (apoE-/-) mice with or without the restoration of macrophage apoE. We used bone marrow transplantation to generate apoE-/- mice with macrophages of 4 genotypes: apoE+/+/ACAT1+/+ (wild type), apoE+/+/ACAT1-/- (ACAT-/-), apoE-/-/ACAT1+/+ (apoE-/-), and apoE-/-/ACAT1-/- (2KO). When macrophage apoE was present, plasma cholesterol levels normalized, and ACAT1 deficiency did not have significant effects on atherogenesis. However, when macrophage apoE was absent, ACAT1 deficiency increased atherosclerosis and apoptosis in the proximal aorta. Cholesterol efflux to apoA-I was significantly reduced (30% to 40%; P<0.001) in ACAT1-/- peritoneal macrophages compared with ACAT1+/+ controls regardless of apoE expression. 2KO macrophages had a 3- to 4-fold increase in ABCA1 message levels but decreased ABCA1 protein levels relative to ACAT1+/+ macrophages. Microarray analyses of ACAT1-/- macrophages showed increases in proinflammatory and procollagen genes and decreases in genes regulating membrane integrity, protein biosynthesis, and apoptosis. Deficiency of macrophage ACAT1 accelerates atherosclerosis in hypercholesterolemic apoE-/- mice but has no effect when the hypercholesterolemia is corrected by macrophage apoE expression. However, ACAT1 deletion impairs ABCA1-mediated cholesterol efflux in macrophages regardless of apoE expression. Changes in membrane stability, susceptibility to apoptosis, and inflammatory response may also be important in this process.

Species‐Specific Regulation of the α‐2(I) Procollagen Gene by Proximal Promoter Elements

Transcriptional regulation of the human alpha 2(I) procollagen proximal promoter involves the interaction of trans-acting factors at the inverted CCAAT box (G/CBE) located at position -80 and an adjacent GGAGGCCC-box at -70. Both these elements have previously been shown to be essential for activity of the human promoter. This study investigated nucleotide differences at three sites (-74, -72 and -71) between the human and mouse promoters that were sufficient to abolish trans-acting factor binding with the mouse sequence (GGAGACGT). Two distinct DNA-protein interactions were detected on the human -107/+54 promoter fragment while a single interaction was observed at the equivalent mouse promoter. One of these factors is the CCAAT-binding factor (CBF) and it's binding was observed on both the human and mouse promoters. Although the GGAGGCCC DNA-binding element was not detected on the mouse promoter, GGAGGCC-binding proteins were present in mouse nuclear extracts as observed by their interaction with the human promoter. Functional analysis of the human and mouse -343/+54 and -107/+54 promoter regions revealed significant differences between species; the human constructs having higher activity than the mouse. The differences in promoter activity between species may in part be a result of the nucleotide differences in the GGAGGCCC-box. Mutations in this region of the human -107/+54 promoter prevented DNA-protein interaction and lowered promoter activity. These results support the hypothesis that the GGAGGCCC-box in the human alpha 2(1) procollagen promoter has a regulatory function and that there exists a species-specific difference in transcription factor binding and regulation of the gene.

Hepatic Gene Expression in Protoporphyic Fech Mice Is Associated with Cholestatic Injury but Not a Marked Depletion of the Heme Regulatory Pool

BALB/c Fech(m1Pas) mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism (Alas1 and Hmox1, respectively) were only modestly affected even in the presence of the cytochrome P450-inducing CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. In contrast, in BALB/c mice exhibiting griseofulvin-induced hepatic protoporphyria with induction and destruction of cytochrome P450, both Alas1 and Hmox1 genes were markedly up-regulated. Other expression profiles in BALB/c Fech(m1Pas) mice identified roles for oxidative mechanisms in liver injury while modulated gene expression of hepatocyte transport proteins and cholesterol and bile acid synthesis illustrated the development of cholestasis. Subsequent inflammation and cirrhosis were also shown by the up-regulation of cytokine, cell cycling, and procollagen genes. Thus, gene expression profiles studied in Fech(m1Pas) mice may provide candidates for human polymorphisms that explain the sporadic hepatic consequences of erythropoietic protoporphyria.

Procollagen type I gene expression and cell proliferation are increased in lipodermatosclerosis

Lipodermatosclerosis (LDS) is characterized by a hardening and hyperpigmentation of lower leg skin as a consequence of chronic venous insufficiency. The degree of skin hardening or fibrosis associated with LDS is proposed to relate directly to skin breakdown and venous ulcer formation as well as to a subsequent delay in ulcer healing. To determine whether elevated procollagen type I gene expression and increased cell proliferation are responsible for the fibrotic changes associated with LDS. Skin biopsies were obtained from the legs of patients with varying degrees of chronic venous disease and were assessed for procollagen gene expression by in-situ hybridization and for cell proliferation by immunolocalization of proliferating cell nuclear antigen. The number of cells expressing procollagen type I mRNA (COL1A1) was significantly higher in the dermis of LDS-affected skin compared with samples from the other patient groups. In addition, there was a significant increase in the number of dermal fibroblasts undergoing proliferation in both LDS samples and skin samples prior to LDS changes compared with control samples. However, there was no significant difference in level of inflammation in biopsy samples between patient classes. These results suggest that enhanced cell proliferation and procollagen gene expression are both involved in LDS development. Furthermore, fibrotic changes may occur in the absence of, or subsequent to, any significant inflammatory response, indicating that additional profibrotic factors produced in the skin as a consequence of chronic venous insufficiency may play a role in LDS formation.

Transcriptional profiling of early onset diet-induced atherosclerosis in apolipoprotein E-deficient mice

Excessive dietary fat and cholesterol exacerbate atherosclerosis. To obtain unbiased insight into the early pathological changes induced by fat feeding in the artery wall, we used high-density microarrays to generate transcriptional profiles of aortic tissue from two groups of atherosclerosis-prone apolipoprotein E-null mice: controls maintained on standard chow and experimental animals exposed short-term to a Western-type diet, a regimen which produced severe hypercholesterolemia without significant development of atheromas. By applying rigorous selection criteria, we identified 311 genes differentially regulated by these dietary conditions. The set of diet-regulated genes exhibited striking functional relationships and represented both novel and known regulatory networks implicated in injury of the artery wall, including cell adhesion genes, histocompatibility antigen and major histocompatibility complex genes, flavin-containing monooxygenases, interferon-regulated genes, small inducible cytokines, collagen and procollagen genes, and complement system components. Further examination of genes identified by this study will provide insights into the molecular mechanisms by which high-fat cholesterol-rich dietary regime initiates pathological alterations in healthy arteries.

CXCL11 Attenuates Bleomycin-induced Pulmonary Fibrosis via Inhibition of Vascular Remodeling

Aberrant vascular remodeling is a central hallmark for the development and progression of idiopathic pulmonary fibrosis. The mechanisms underlying the pathophysiologic alterations, however, are poorly understood. A recent phase II trial of interferon gamma-1b has demonstrated a trend toward a decrease in profibrotic and proangiogenic biologic markers, and upregulation of lung CXCL11 mRNA and bronchoalveolar lavage fluid and plasma protein levels of CXCL11. We hypothesized that net aberrant vascular remodeling seen during the pathogenesis of fibroplasia and deposition of extracellular matrix during bleomycin-induced pulmonary fibrosis can be attenuated by treatment with the angiostatic ELR(-) CXC chemokine, CXCL11. In a preclinical model, systemic administration of CXCL11 reduced pulmonary collagen deposition, procollagen gene expression, and histopathologic fibroplasia and extracellular matrix deposition in the lung of bleomycin-treated mice. CXCL11 treatment significantly reduced bleomycin-induced pulmonary fibrosis without altering specific lung leukocyte populations. CXCR3 is not expressed on fibroblasts and CXCL11 had no direct functional effect on pulmonary fibroblasts. The angiogenic activity in the lung was significantly decreased, however, and CXCL11 treatment reduced the total number of endothelial cells in the lung following bleomycin exposure. The results suggest that CXCL11 inhibits pulmonary fibrosis by altering aberrant vascular remodeling.

Solar Ultraviolet Irradiation Reduces Collagen in Photoaged Human Skin by Blocking Transforming Growth Factor-β Type II Receptor/Smad Signaling

Ultraviolet (UV) irradiation from the sun reduces production of type I procollagen (COLI), the major structural protein in human skin. This reduction is a key feature of the pathophysiology of premature skin aging (photoaging). Photoaging is the most common form of skin damage and is associated with skin carcinoma. TGF-beta/Smad pathway is the major regulator of type I procollagen synthesis in human skin. We have previously reported that UV irradiation impairs transforming growth factor-beta (TGF-beta)/Smad signaling in mink lung epithelial cells. We have investigated the mechanism of UV irradiation impairment of the TGF-beta/Smad pathway and the impact of this impairment on type I procollagen production in human skin fibroblasts, the major collagen-producing cells in skin. We report here that UV irradiation impairs TGF-beta/Smad pathway in human skin by down-regulation of TGF-beta type II receptor (TbetaRII). This loss of TbetaRII occurs within 8 hours after UV irradiation and precedes down-regulation of type I procollagen expression in human skin in vivo. In human skin fibroblasts, UV-induced TbetaRII down-regulation is mediated by transcriptional repression and results in 90% reduction of specific, cell-surface binding of TGF-beta. This loss of TbetaRII prevents downstream activation of Smad2/3 by TGF-beta, thereby reducing expression of type I procollagen. Preventing loss of TbetaRII by overexpression protects against UV inhibition of type I procollagen gene expression in human skin fibroblasts. UV-induced down-regulation of TbetaRII, with attendant reduction of type I procollagen production, is a critical molecular mechanism in the pathophysiology of photoaging.

Syndromes with congenital brittle bones.

BackgroundThere is no clear definition of osteogenesis imperfecta (OI). The most widely used classification of OI divides the disease in four types, although it has been suggested that there may be at least 12 forms of OI. These forms have been named with numbers, eponyms or descriptive names. Some of these syndromes can actually be considered congenital forms of brittle bones resembling OI (SROI).DiscussionA review of different syndromes with congenital brittle bones published in the literature is presented. Syndromes are classified in "OI" (those secondary to mutations in the type I pro-collagen genes), and "syndromes resembling OI" (those secondary to mutations other that the type I pro-collagen genes, identified or not). A definition for OI is proposed as a syndrome of congenital brittle bones secondary to mutations in the genes codifying for pro-collagen genes (COL1A1 and COL1A2).SummaryA debate about the definition of OI and a possible clinical and prognostic classification are warranted.

Effect of cytokine and sandostatin on collagen synthesis and expression of procollagen mRNA by lens epithelial cells

To study the effects of epidermal growth factor (EGF), interleukin-1 (IL-1), interferon-gamma (IFN-gamma) and sandostatin on collagen synthesis and expression of types I and III procollagen mRNA by bovine lens epithelial cells in vitro. Cells were plated in 96-well tissue culture plates. After attached, serum-free medium containing EGF 10(-2) - 10(2) ng/ml, IL-1 10 - 10(5) ng/ml, sandostatin 10(-11) - 10(-7) mol/L and IFN-gamma 10 - 10(5) U/ml were added into the plates. Collagen synthesis was measured with (3)H-proline incorperation, and expression of procollagen mRNA was detected with Northern blot. EGF 10(-1) - 10(2) ng/ml and IL-1 10(2) - 10(5) ng/ml obviously promoted collagen synthesis. EGF 1 ng/ml and IL-1 10(3) ng/ml increased the expression of types I and III procollagen mRNA. IFN-gamma 10(3) - 10(5) ng/ml and sandostatin 10(-10) - 10(-7) mol/L decreased collagen synthesis. IFN-gamma 10(3) U/ml and sandostatin 10(-9) mol/L decreased the expression of types I and III procollagen mRNA. EGF and IL-1 may enhance the occurrence of posterior capsular opacification, they affect types I and III procollagen gene expression at transcription level. IFN-gamma and sandostatin might be used to prevent the occurrence of posterior capsular opacification.

Knocking out the bad allele.

Knocking out the bad allele.

Effect of hyperbaric oxygen on the ligament healing process in rats.

Animal experiments were done to investigate whether administration of hyperbaric oxygen promotes scar tissue formation, increases expression of the Type I procollagen gene, and improves the tensile properties of healing ligament. In 76 Sprague-Dawley rats, a 2-mm segment of the medial collateral ligament was removed. Thirty-eight rats were exposed to hyperbaric oxygen at 2.5 atmospheres absolute for 2 hours 5 days per week (Group H), whereas the remaining rats were exposed to room air (Group C). The animals were sacrificed at 3, 7, 14, and 28 days postoperatively. In situ hybridization histochemistry was done to examine the Type I procollagen gene expression in healing ligaments in 40 rats, whereas a tensile failure test was done in the remaining rats. The amount of scar tissue was greater in Group H than in Group C. Type I procollagen gene expression at 7 or 14 days was significantly greater in Group H than in Group C. The ultimate load and stiffness in Group H were significantly greater than in Group C at 14 days. Administration of hyperbaric oxygen promotes scar tissue formation and increases Type I procollagen gene expression in healing ligaments. These effects are associated with the improvement of their tensile properties.

Signaling “cross-talk” between TGF-β1 and ECM signals in chondrocytic cells

The objective of this investigation was to clarify how the integrin pathway modulates downstream effectors of the TGF-β1 pathway in chondrocytic cell signaling. The levels of Smad2 and Smad3 phosphorylation upon TGF-β1 or α2β1 integrin (Type II collagen) stimulation were analyzed by Western blotting techniques. Cellular response was determined by quantitation of procollagen gene expression. Stimulation of cells with TGF-β1 and Type II collagen led to rapid phosphorylation of Smad2 and 3 with phosphorylation peaking between 15 min and 1 h. Combined stimulation led to a synergistic increase in the phosphorylation of Smad2 and Smad3. Type II collagen gene expression paralleled Smad phosphorylation. Type II collagen modulates the TGF signaling cascade involving Smad2 and Smad3 leading to an increase in Type II collagen transcription. Therefore, we conclude that TGF-β1 and integrin stimuli interact prior to Smad2 and 3 phosphorylation in the cytoplasm of chondrocytic cells and regulates the expression of ECM components in chondrocytes.

B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts

We investigated the role of B-Myb, a cell-cycle-regulated transcription factor, in the expression of the alpha1 (I) pro-collagen gene (COL1A1) in scleroderma fibroblasts. Scleroderma or SSc (systemic sclerosis) is a fibrotic disease characterized by excessive production of extracellular matrix components, especially type I collagen. Northern-blot analysis showed an inverse relationship between COL1A1 mRNA expression and that of B-Myb during exponential cell growth and during quiescence in human SSc fibroblasts. Overexpression of B-Myb in SSc fibroblasts was correlated with decreased COL1A1 mRNA expression. Transient transfections localized the down-regulatory effect of B-Myb to a region containing the proximal 174 bp of the COL1A1 promoter that does not contain B-Myb consensus binding sites. Gel-shift analysis, using nuclear extracts from normal and SSc fibroblasts transfected with B-Myb, showed no differences in DNA-protein complex formation when compared with the nuclear extracts from mock-transfected cells. However, we found that B-Myb decreases Sp1 (specificity protein 1) and CBF (CCAAT-binding factor) binding for their specific sites localized in the 174 bp COL1A1 proximal promoter. These results were also confirmed using B-Myb-immunodepleted nuclear extracts. Furthermore, immunoprecipitation assays using SSc nuclear extracts demonstrated a physical interaction of B-Myb with Sp1 and CBF transcription factors, and also an interaction between Sp1 and CBF. In addition, by employing full-length or deleted B-Myb cDNA construct, we found that B-Myb down-regulates the COL1A1 proximal promoter through its C-terminal domain. Thus these results suggest that B-Myb may be an important factor in the pathway(s) regulating collagen production in SSc fibroblasts.

Differential roles of extracellular signal-regulated kinase 1/2 and p38MAPK in mechanical load-induced procollagen alpha1(I) gene expression in cardiac fibroblasts.

We have previously demonstrated that mechanical loading of cardiac fibroblasts leads to increased synthesis and gene expression of the extracellular matrix protein collagen. We hypothesised that the upregulation of procollagen gene expression in cardiac fibroblasts, in response to cyclic mechanical load, is mediated by one or more members of the MAP kinase family. To test this hypothesis, the effect of mechanical load on the activation of extracellular signal-regulated kinase (ERK) 1/2, p46/54JNK, and p38MAPK was examined in rat cardiac fibroblasts. Peak phosphorylation of ERK 1/2, p38MAPK kinases, and p46/54JNK was observed following 10-20 min of continuous cyclic mechanical load. Mechanical load significantly increased procollagen alpha1(I) mRNA levels up to twofold above static controls after 24 h. This increase was completely abolished by the MEK 1/2 inhibitor U0126, with no effect on basal levels. In contrast, SB203580, a specific inhibitor of p38MAPK, enhanced both basal and stretch-stimulated levels of procollagen mRNA. Consistent with this finding, selective activation of the p38MAPK signalling pathway by expression of MKK6(Glu), a constitutive activator of p38MAPK, significantly reduced procollagen alpha1(I) promoter activity. SB203580-dependent increase in procollagen alpha1(I) was accompanied by ERK 1/2 activation, and inhibition of this pathway completely prevented SB203580-induced procollagen alpha1(I) expression. These results suggest that mechanical load-induced procollagen alpha1(I) gene expression requires ERK 1/2 activation and that the p38MAPK pathway negatively regulates gene expression in cardiac fibroblasts. These pathways are likely to be key in events leading to matrix deposition during heart growth and remodelling induced by mechanical load.

Construction of specific ribozymes against second exon of α1 I and α1 III procollagen genes

目的 构建出针对α1Ⅰ型及α1Ⅲ型前胶原基因第2外显子(Exon 2)片段的核酶.方法根据α1Ⅰ型及α1Ⅲ型前胶原基因的碱基序列以及锤头型核酶(ribozyme)的结构域要求,利用聚合酶链反应(PCR)分别构建了针对各自的Exon 2 区域的核酶基因,并克隆到T载体(pGEM-T vector)T7启动子下游.结果分别以引物对PⅠL/PⅠR及PⅢL/PⅢR进行PCR扩增,经琼脂糖凝胶电泳各得到约62bp的DNA片段,与预期的核酶基因大小一致.结论经PCR限制性内切酶酶切及序列测定分析,证实为所设计的插入正确的核酶基因,从而为大量制备核酶及其体外切割实验和胞内与mRNA相互作用的研究打下了基础。

Transcriptional regulation of human alpha1(I) procollagen gene in dermal fibroblasts.

To clarify the fractional activity of promoters from human alpha1(I) procollagen gene, the interaction between cis-elements and consensus DNA-binding proteins responsible for high promoter activity, and the potential application of promoter competitors as well as cytokines for antifibrogenesis. Sequence between 2483 bp upstream of the start of transcription and 42 bp downstream of this site was investigated with serial 5'-deletion. The 5'-deleted promoters recombined with chloramphenicol acetyltransferase (CAT) as reporter gene were transiently transfected to human dermal fibroblasts. Electrophoretic mobility shift assay was performed to show the DNA-protein binding capacity of the promoter sequence. Cytokines including tumor necrosis factor alpha (TNFalpha) and interferons (INFs) were added to the culture medium of transiently transfected fibroblasts. Competitor DNA for the binding sites of Sp-1, Ap-1 and NF-1 was individually cotransfected transiently in order to block the promoter-driven CAT expression. Sequences of -2483 to +42 bp and -268 to +42 bp of human alpha1(I) procollagen gene had high activity as promoters. Binding sites for Ap-1 and Sp-1 were among the cis-regulatory elements recognizing consensus transcription factors responsible for basal promoter activity of sequence -268 to +42 bp. TNFalpha, IFNalpha, IFNbeta showed inhibitory effects on sequence -2 483 to +42 bp as promoter with activities 43%, 62% and 60% of control respectively. Transfection of the promoter competitors could reverse the promoter activity of -268 to +42 bp 40-60%. Sequences of -2 483 to +42 bp recombined with reporter gene provide an ideal construction for transcriptional study of alpha1(I) procollagen gene. The anti-collagen capacity of TNFalpha and IFNs is associated with their transcriptional regulation. Ap-1 and Sp-1 mediate the basal transcriptional activation of human alpha1(I) procollagen gene in dermal fibroblasts. Competitors for highly active promoters might be a novel potential candidate in fibrotic blockade.

Interferon-γ Interferes with Transforming Growth Factor-β Signaling through Direct Interaction of YB-1 with Smad3

Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) exert antagonistic effects on collagen synthesis in human dermal fibroblasts. We have recently shown that Y box-binding protein YB-1 mediates the inhibitory effects of IFN-gamma on alpha2(I) procollagen gene (COL1A2) transcription through the IFN-gamma response element located between -161 and -150. Here we report that YB-1 counter-represses TGF-beta-stimulated COL1A2 transcription by interfering with Smad3 bound to the upstream sequence around -265 and subsequently by interrupting the Smad3-p300 interaction. Western blot and immunofluorescence analyses using inhibitors for Janus kinases or casein kinase II suggested that the casein kinase II-dependent signaling pathway mediates IFN-gamma-induced nuclear translocation of YB-1. Down-regulation of endogenous YB-1 expression by double-stranded YB-1-specific RNA abrogated the transcriptional repression of COL1A2 by IFN-gamma in the absence and presence of TGF-beta. In transient transfection assays, overexpression of YB-1 in human dermal fibroblasts exhibited antagonistic actions against TGF-beta and Smad3. Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smad-binding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-gamma/YB-1 counteracts TGF-beta/Smad3. They also indicate that IFN-gamma/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-gamma response element and through a cross-talk with the TGF-beta/Smad signaling pathway.