COL1A2 Promoter Research Articles

THU0033 SET7/9 and SIRT1 induce promoter-driven collagen 2ALPHA1 expression in 3D cultured human chodrocytes

BackgroundOsteoarthritis (OA) is characterized by disrupted balance between extracellular matrix (ECM) synthesis and breakdown, leading to loss of articular cartilage (AC) and severe joint pain-dysfunction. Collagen type IIα1 (Col2α1) is...

Overexpression of Cdk6 and Ccnd1 in chondrocytes inhibited chondrocyte maturation and caused p53-dependent apoptosis without enhancing proliferation

Cell proliferation and differentiation are closely coupled. However, we previously showed that overexpression of cyclin-dependent kinase (Cdk6) blocks chondrocyte differentiation without affecting cell-cycle progression in vitro. To investigate whether Cdk6 inhibits chondrocyte differentiation in vivo, we generated chondrocyte-specific Cdk6 transgenic mice using Col2a1 promoter. Unexpectedly, differentiation and cell-cycle progression of chondrocytes in the Cdk6 transgenic mice were similar to those in wild-type mice. Then, we generated chondrocyte-specific Ccnd1 transgenic mice and Cdk6/Ccnd1 double transgenic mice to investigate the possibility that Cdk6 inhibits chondrocyte differentiation through E2f activation. Bromodeoxyuridine (BrdU)-positive chondrocytes and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive chondrocytes were increased in number, and chondrocyte maturation was inhibited only in Cdk6/Ccnd1 transgenic mice (K6(H)/D1(H) mice), which showed dwarfism. Retinoblastoma protein (pRb) was highly phosphorylated but p107 was upregulated, and the expression of E2f target genes was dysregulated as shown by upregulation of Cdc6 but downregulation of cyclin E, dihydrofolate reductase (dhfr), Cdc25a and B-Myb in chondrocytes of K6(H)/D1(H) mice. Similarly, overexpression of Cdk6/Ccnd1 in a chondrogenic cell line ATDC5 highly phosphorylated pRb, upregulated p107, induced apoptosis, upregulated Cdc6 and downregulated cyclin E, dhfr and B-Myb and p107 small interfering RNA reversed the expression of downregulated genes. Further, introduction of kinase-negative Cdk6 and cyclin D1 abolished all effects by Cdk6/cyclin D1 in ATDC5 cells, indicating the requirement of the kinase activity on these effects. p53 deletion partially restored the size of the skeleton and almost completely rescued chondrocyte apoptosis, but failed to enhance chondrocyte proliferation in K6(H)/D1(H) mice. These findings indicated that Cdk6/Ccnd1 overexpression inhibited chondrocyte maturation and enhanced G1/S cell-cycle transition by phosphorylating pRb, but the chondrocytes failed to accomplish the cell cycle, and underwent p53-dependent apoptosis probably due to the dysregulation of E2f target genes. Our findings also indicated that p53 deletion in addition to the inactivation of Rb was not sufficient to accelerate chondrocyte proliferation, suggesting the resistance of chondrocytes to sarcomagenesis.

Abstract 665: Promoter DNA methylation is associated with metastatic properties of pancreatic cancer.

Abstract The inability of early detection of pancreatic tumors is largely due to a lack of biomarkers and a poor understanding of molecular mechanisms conferring carcinogenic dissemination. Recent evidence has indicated that various neoplastic phenotypes are governed by epigenomic alterations (DNA methylation or histone modifications) which confer reversible effects without changing nucleotide sequences themselves. During the past years, we have compared DNA methylation profiles between the parental pancreatic cells FG and its isogenic variant L3.6pl that gained remarkable migratory and invasive properties in culture (33- and 20-fold increase) and in mice (Neoplasia; 1(1); page 50, 1999). DNA methylation profiling was conducted by a method known as MBD-isolated Genome Sequencing which captures methylated DNA via a methyl-CpG binding protein followed by genome-wide DNA sequencing using Illumina sequencer (GAII). Reads were then aligned to a reference human genome browser (HG18) whereby the abundance of read coverage was calculated and indicative of methylation levels occurring at given loci. These methylation profiles were used to generate a list of candidate genes that displayed higher levels of DNA methylation within the L3.6 compared to FG cell lines. We hypothesize that these hypermethylated loci may be correlated to the enhanced invasive properties associated with not only L3.6pl, but also other metastatic pancreatic carcinomas in general. Among these candidates, COL7A1 was of interest. It encodes for a basement membrane protein that is known to be a type VII collagen and is the main component of anchoring fibrils that are involved in cellular processes such as cell adhesion and motility. Silenced expression of COL7A1 by DNA methylation has been shown by two lines of evidence generated from our laboratory. (1) CpG islands situated within COL7A1 promoter displayed prominent promote activity. (2) The induction of methylation by an in vitro method impaired its transcriptional activity. To determine if other epigenetic alterations such as histone modifications led to a similar reduction of COL7A1 expression, a histone deacetylase inhibitor, Trichostatin A, was introduced. It was found that this agent was unable to restore similar expression levels of COL7A1 compared to treatment of de-methylation agent (5-azacytidine). These data suggest that DNA methylation is an important mechanism governing COL7A1 expression We further suggest that this aberration may serve as a general phenomenon, as COL7A1 was concordantly methylated in other metastatic pancreatic cell lines including ASPC-1, Capan-2, and Miacapa-2. Taken together, we hypothesize that promoter methylation leads to a reduced COL7A1 expression followed by an induction of metastasis in pancreatic adenocarcinoma. Thus, COL7A1 promoter could potentially serve as a diagnostic marker and a therapeutic target for impeding metastasis. Citation Format: Huey-Jen L. Lin, Zhengang Peng, Amanda Fisher, Jennifer C. Weber, Ying-Wei Li. Promoter DNA methylation is associated with metastatic properties of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 665. doi:10.1158/1538-7445.AM2013-665

Transforming Growth Factor β1 Induces the Expression of Collagen Type I by DNA Methylation in Cardiac Fibroblasts

Transforming growth factor-beta (TGF-β), a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1) expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs) were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

Creating conditional knockout mice for OA research

s / Osteoarthritis and Cartilage 21 (2013) S1–S8 S7 I-22 CREATING CONDITIONAL KNOCKOUT MICE FOR OA RESEARCH P.J. Roughley. Shriners Hosp., Montreal, QC, Canada Purpose: Traditional means for creating OA in animal models has involved the use of surgery to destabilize the joint or the creation of joint inflammation, both of which result in subsequent cartilage degeneration. However, while these methods allow the progression of cartilage degeneration to be studied, they do not permit the necessity of individual molecules to be established. The use of knockout mice can potentially solve this problem. However, global knockouts are often not useful as they may have lethal consequences during development due to the involvement of multiple organs. This led to the development of conditional knockout mice, in which gene deletion is confined to cartilage. Even cartilage-specific knockout can be problematic, when the gene of interest affects chondrogenesis or growth, but this can be overcome by the use of inducible conditional knockout mice. This workshop will describe how such mice can be generated. Methods: Gene knockout is based on the ability to excise genomic regions that are essential for protein expression or function, and the Cre/LoxP system is most commonly used for this purpose. This involves the creation of a Floxed mouse line by the insertion of LoxP sites around the gene region of interest, and the subsequent breeding with a mouse line expressing Cre recombinase to excise at the LoxP sites and then recombine the excised gene. Cartilage-specificity is achieved having Cre expression under control of a cartilage specific gene promoter, such as that for type II collagen (Col2a1). For additional temporal selectivity, Cre expression is also placed under control of either doxycycline or tamoxifen administration. With this system, knockout can be achieved at any time throughout life, as long as the promoter driving Cre expression remains active. Results: Cartilage-specific knockout was initially achieved using the Col2a1 promoter to drive Cre expression, which allows gene knockout in both the articular cartilages of all diathrodial joints and the intervertebral discs of the spine. Twomodifications to this system have been developed to facilitate inducible Cre expression. In the first, Cre is expressed as a fusion protein in conjunctionwith the estrogen receptor, and is only translocated to the nucleus upon tamoxifen administration. In the second, Cre expression is placed under control of rtTA expression which in turn is driven by the Col2a1 promoter, with rtTA function being dependent upon doxycycline administration. Use of the Col2a1 promoter is, however, limited to early adult life as the expression of this promoter declines with age. This limitation has been overcome in two systems by use of the aggrecan promoter or enhancer. In one system, the Cre/estrogen receptor cassette has been place within the 30-UTR of the endogenous aggrecan gene, and in the other, the aggrecan enhancer has been used to ensure prolonged activity of the Col2a1 promoter. Conclusions: Conditional knockout mice provide a means of studying the participation of individual molecules in the progression of cartilage degeneration, and when coupled with temporal selectivity this can be performed at any stage of the disease process. The knockoutmice can be used directly or in combination with traditional surgical or biochemical intervention to induce the OA process. I-23 IMPACT OF INJURY ON JOINT STRUCTURE R.B. Frobell. Orthopedics, Clinical Sci., Lund Univ., Lund, Sweden Purpose: Knee trauma is a well-recognized and established risk factor for OA development in the longer term. The purpose of this presentation is to give an overview of trauma related structural injuries and change possibly involved the early OA disease process. Methods: Using MR imaging techniques and biochemical analysis in longitudinal trials starting in the acute phase after injury, we have monitored the structural and biochemical response of injury with the aim of identifying early changes possibly related to subsequent development of OA. Results: This presentation will give an overview of the current publications of the structural and biochemical response to knee trauma and present possible early signs of change that may be related to subsequent development of OA. Conclusions: Early changes of bone shape and cartilage thickness occur after ACL injury although the relation to later OA development remains to be determined. There also seems to be evidence in support of a biochemical alterations in the early phase of injury however, few studies are longitudinal and even fewer have correlated biochemical and structural change in this very early phase of OA. I-24 EMERGING PARADIGM: ROLES OF INFLAMMATION IN OA R. Terkeltaub y,z. yUniv. of California, San Diego, CA, USA; zVAMC, San

The Rescue of Dentin Matrix Protein 1 (DMP1)-deficient Tooth Defects by the Transgenic Expression of Dentin Sialophosphoprotein (DSPP) Indicates That DSPP Is a Downstream Effector Molecule of DMP1 in Dentinogenesis

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

Local Regulation of Tooth Mineralization by Sphingomyelin Phosphodiesterase 3

Sphingomyelin phosphodiesterase 3 (Smpd3) encodes a membrane-bound enzyme that cleaves sphingomyelin to generate several bioactive metabolites. A recessive mutation called fragilitas ossium (fro) in the Smpd3 gene leads to impaired mineralization of bone and tooth extracellular matrix (ECM) in fro/fro mice. In teeth from fro/fro mice at various neonatal ages, radiography and light and electron microscopy showed delayed mantle dentin mineralization and a consequent delay in enamel formation as compared with that in control +/fro mice. These tooth abnormalities progressively improved with time. Immunohistochemistry showed expression of SMPD3 by dentin-forming odontoblasts. SMPD3 deficiency, however, did not affect the differentiation of these cells, as shown by osterix and dentin sialophosphoprotein expression. Using a transgenic mouse rescue model (fro/fro; Col1a1-Smpd3) in which Smpd3 expression is driven by a murine Col1a1 promoter fragment active in osteoblasts and odontoblasts, we demonstrate a complete correction of the tooth mineralization delays. In conclusion, analysis of these data demonstrates that Smpd3 expression in odontoblasts is required for tooth mineralization.

In Vivo Overexpression of Tissue-Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin

Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl⁻/⁻ mice) leads to hypophosphatasia, characterized by rickets/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alpl⁻/⁻ mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl⁻/⁻ mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast-specific Col1a1 promoter (Col1a1-Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1-Tnap are 10 to 20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by micro-computed tomography (µCT) analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alpl⁻/⁻ mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1-Tnap] mice mineralize better than nontransgenic controls and osteoblasts from [Col1a1-Tnap⁺/⁻; Alpl⁻/⁻] mice are able to mineralize to the level of Alpl⁺/⁻ heterozygous osteoblasts, whereas Alpl⁻/⁻ osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl⁻/⁻ mice are comprised of phosphorylated forms of OPN whereas wild-type (WT) and [Col1a1-Tnap⁺/⁻; Alpl⁻/⁻] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more dephosphorylated than nontransgenic control cells. Titanium dioxide-liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alpl⁻/⁻ bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S¹⁷⁴FQVS¹⁷⁸DEQY¹⁸²PDAT¹⁸⁶DEDLT¹⁹¹)SHMK and FRIp(S²⁹⁹HELES³⁰⁴S³⁰⁵S³⁰⁶S³⁰⁷)EVN, with one nonlocalized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the promineralization role of TNAP may be related not only to its accepted pyrophosphatase activity but also to its ability to modify the phosphorylation status of OPN.

The transcription factor activating enhancer‐binding protein epsilon (AP–2ε) regulates the core promoter of type II collagen (COL2A1)

The transcription factor activating enhancer-binding protein epsilon (AP-2ε) was recently shown to be expressed during late chondrocyte differentiation, especially in hypertrophic chondrocytes. In this study, we were able to reveal that the promoter of the type II collagen (COL2A1) gene, encoding the extracellular matrix protein type II collagen, is specifically regulated by AP-2ε. Expression of COL2A1 is downregulated at the transition of chondroblasts into hypertrophic chondrocytes and our data provide evidence that AP-2ε is involved in this process. In reporter gene assays, overexpression of AP-2ε in cartilaginous cell lines resulted in a significant reduction in COL2A1 core promoter activity of ~ 45%. Furthermore, we found that this process is dose-dependent and highly specific for the epsilon isoform. Computational analysis offered only a single putative AP-2-binding motif within the chosen promoter fragment but site-directed mutagenesis revealed this motif to be regulatory inactive. After expanding our screening to motifs containing minor differences from the classical AP-2 consensus sequence (5'-GCCN3 GGC-3'), we determined the sequence 5'-GCCCAGGC-3' ranging from position -128 to -135 bp as an important regulatory site and responsible for COL2A1 downregulation through AP-2ε. Interaction of AP-2ε with the COL2A1 promoter at this site was confirmed by chromatin immunoprecipitation and electromobility shift assay. Further, our experiments suggest that at least one additional factor is involved in this process. This is the first study to prove regulation of COL2A1 by AP-2ε highlighting the role of the transcription factor as a modulator of cartilage development.

Conditional Inactivation of TNFα-Converting Enzyme in Chondrocytes Results in an Elongated Growth Plate and Shorter Long Bones

TNFα-converting enzyme (TACE) is a membrane-bound proteolytic enzyme with essential roles in the functional regulation of TNFα and epidermal growth factor receptor (EGFR) ligands. Previous studies have demonstrated critical roles for TACE in vivo, including epidermal development, immune response, and pathological neoangiogenesis, among others. However, the potential contribution of TACE to skeletal development is still unclear. In the present study, we generated a Tace mutant mouse in which Tace is conditionally disrupted in chondrocytes under the control of the Col2a1 promoter. These mutant mice were fertile and viable but all exhibited long bones that were approximately 10% shorter compared to those of wild-type animals. Histological analyses revealed that Tace mutant mice exhibited a longer hypertrophic zone in the growth plate, and there were fewer osteoclasts at the chondro-osseous junction in the Tace mutant mice than in their wild-type littermates. Of note, we found an increase in osteoprotegerin transcripts and a reduction in Rankl and Mmp-13 transcripts in the TACE-deficient cartilage, indicating that dysregulation of these genes is causally related to the skeletal defects in the Tace mutant mice. Furthermore, we also found that phosphorylation of EGFR was significantly reduced in the cartilage tissue lacking TACE, and that suppression of EGFR signaling increases osteoprotegerin transcripts and reduces Rankl and Mmp-13 transcripts in primary chondrocytes. In accordance, chondrocyte-specific abrogation of Egfr in vivo resulted in skeletal defects nearly identical to those observed in the Tace mutant mice. Taken together, these data suggest that TACE-EGFR signaling in chondrocytes is involved in the turnover of the growth plate during postnatal development via the transcriptional regulation of osteoprotegerin, Rankl, and Mmp-13.

Osterix and Runx2 cooperate to induce expression of the Col1a1 promoter

Osterix and Runx2 cooperate to induce expression of the Col1a1 promoter

Osterix induces Col1a1 gene expression through binding to Sp1 sites in the bone enhancer and proximal promoter regions

Bone-specific transcription factors promote differentiation of mesenchymal precursors toward the osteoblastic cell phenotype. Among them, Runx2 and Osterix have been widely accepted as master osteogenic factors, since neither Runx2 nor Osterix null mice form mature osteoblasts. Recruitment of Osterix to a number of promoters of bone-specific genes has been shown. However, little is known about the functional interactions between Osterix and the Col1a1 promoter. In this study we determined in several mesenchymal and osteoblastic cell types that either BMP-2 or Osterix overexpression increased Col1a1 transcription. We identified consensus Sp1 sequences, located in the proximal promoter and in the bone-enhancer, as Osterix binding regions in the Col1a1 promoter in vitro and in vivo. Furthermore, we show that p38 or Erk MAPK signaling is required for maximal transcriptional effects on Col1a1 expression. Runx2 has been shown to activate Col1a1 expression through binding to sites which are located close to the Sp1 sites where Osterix binds. Our data show that overexpression of Runx2 and Osterix leads to a cooperative effect on the expression of the Col1a1 endogenous gene and its promoter reporter construct. These effects mainly affect the long isoform of Osterix which suggest that the two Osterix isoforms might display some differential effects on the transactivation of bone-specific genes.

SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells

Decreased expression of collagen by vascular smooth muscle cells (SMCs) within the atherosclerotic plaque contributes to the thinning of the fibrous cap and poses a great threat to plaque rupture. Elucidation of the mechanism underlying repressed collagen type I (COL1A2) gene would potentially provide novel solutions that can prevent rupture-induced complications. We have previously shown that regulatory factor for X-box (RFX5) binds to the COL1A2 transcription start site and represses its transcription. Here we report that SIRT1, an NAD-dependent, class III deacetylase, forms a complex with RFX5. Over-expression of SIRT1 or NAMPT, which synthesizes NAD+ to activate SIRT1, or treatment with the SIRT1 agonist resveratrol decreases RFX5 acetylation and disrupts repression of the COL1A2 promoter activity by RFX5. On the contrary, knockdown of SIRT1 or treatment with SIRT1 inhibitors induces RFX5 acetylation and enhances the repression of collagen transcription. SIRT1 antagonizes RFX5 activity by promoting its nuclear expulsion and proteasomal degradation hence dampening its binding to the COL1A2 promoter. The pro-inflammatory cytokine IFN-γ represses COL1A2 transcription by down-regulating SIRT1 expression in SMCs. Therefore, our data have identified as novel pathway whereby SIRT1 maintains collagen synthesis in SMCs by modulating RFX5 activity.

Overexpression of H1 calponin in osteoblast lineage cells leads to a decrease in bone mass by disrupting osteoblast function and promoting osteoclast formation

H1 calponin (CNN1) is known as a smooth muscle-specific, actin-binding protein which regulates smooth muscle contractive activity. Although previous studies have shown that CNN1 has effect on bone, the mechanism is not well defined. To investigate the role of CNN1 in maintaining bone homeostasis, we generated transgenic mice overexpressing Cnn1 under the control of the osteoblast-specific 3.6-kb Col1a1 promoter. Col1a1-Cnn1 transgenic mice showed delayed bone formation at embryonic stage and decreased bone mass at adult stage. Morphology analyses showed reduced trabecular number, thickness and defects in bone formation. The proliferation and migration of osteoblasts were decreased in Col1a1-Cnn1 mice due to alterations in cytoskeleton. The early osteoblast differentiation of Col1a1-Cnn1 mice was increased, but the late stage differentiation and mineralization of osteoblasts derived from Col1a1-Cnn1 mice were significantly decreased. In addition to impaired bone formation, the decreased bone mass was also associated with enhanced osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) staining revealed increased osteoclast numbers in tibias of 2-month-old Col1a1-Cnn1 mice, and increased numbers of osteoclasts co-cultured with Col1a1-Cnn1 osteoblasts. The ratio of RANKL to OPG was significantly increased in Col1a1-Cnn1 osteoblasts. These findings reveal a novel function of CNN1 in maintaining bone homeostasis by coupling bone formation to bone resorption.

NF-κB Accumulation Associated with COL1A1 Transactivators Defects during Chronological Aging Represses Type I Collagen Expression through a –112/–61-bp Region of the COL1A1 Promoter in Human Skin Fibroblasts

The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-κB (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-κB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

Constitutive activation of c-Abl/protein kinase C-δ/Fli1 pathway in dermal fibroblasts derived from patients with localized scleroderma

A noncanonical pathway of transforming growth factor-β signalling, the c-Abl/protein kinase C-δ (PKC-δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts. To investigate the significance of the c-Abl/PKC-δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc). The activation status of the c-Abl/PKC-δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet-derived growth factor receptor inhibitor AG1296 and gene silencing of c-Abl on the expression levels of type I collagen were evaluated by immunoblotting. The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c-Abl were elevated compared with cultured normal fibroblasts and PKC-δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c-Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts. Constitutive activation of the c-Abl/PKC-δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc.

Activation ofCOL1A2promoter in human fibroblasts byEscherichia coli

The relationship between bacterial infection and collagen production was investigated using human fibroblasts transfected with the promoter of COL1A2 , which encodes the α1 chain of human type I collagen, linked to a luciferase reporter. The cells were used to assess the gene promoter activity of COL1A2 following bacterial stimulation. The COL1A2 promoter was activated by stimulation with fixed Escherichia coli in a dose-dependent manner, but not by fixed Staphylococcus aureus. Enhancement of collagen production was observed in the E.coli-stimulated fibroblasts compared to those without stimulation. Both anti-human Toll-like receptor (TLR) 4 antibody and polymyxin B clearly blocked the COL1A2 promoter activity stimulated by E.coli, while antibodies against human TLR2 and human transforming growth factor-β (TGF-β) receptor type II did not. These results indicate that E.coli can directly interact with TLR4 expressed on the surface of fibroblasts and can further induce human type I collagen gene expression and collagen production in these cells. These data also suggest that infection by gram-negative bacteria may cause fibrosis.

Synergistic roles of scleraxis and Smads in the regulation of collagen 1α2 gene expression

Cardiac fibrosis is marked by increased deposition of extracellular matrix components including fibrillar collagens, leading to impaired cardiac contractility and function. We recently demonstrated that the transcription factor scleraxis is expressed in collagen-producing cardiac fibroblasts and myofibroblasts, is up-regulated in the collagen-rich scar following myocardial infarction and is sufficient to transactivate the human collagen 1α2 (COL1A2) gene, suggesting a central role in fibrosis. Here we describe the mechanism of scleraxis-mediated regulation of the COL1A2 promoter. Using chromatin immunoprecipitation in primary human cardiac fibroblasts in combination with luciferase assays, we demonstrate that two E box sequences within the proximal COL1A2 promoter are required for scleraxis-mediated transactivation. Expression of scleraxis itself was induced by receptor Smad3, an effector of the pro-fibrotic growth factor TGF-β1, and attenuated by inhibitory Smad7. TGF-β1 augmented the effect of scleraxis on COL1A2 transactivation, an effect which was due to synergy between scleraxis and Smad3. Mutation of the COL1A2 Smad-binding element significantly attenuated the ability of scleraxis to transactivate the promoter, while mutation of the scleraxis-interacting E boxes attenuated the effect of Smad3, suggesting that these factors form a common signaling complex at the promoter. COL1A2 promoter transactivation and Col1α2 gene expression in cardiac fibroblasts were completely abrogated by a scleraxis basic domain deletion mutant in a dominant negative fashion, blocking the ability of TGF-β1 to activate collagen synthesis and suggesting that scleraxis–DNA interaction is absolutely required for this process. Scleraxis thus appears to play a key role in the transcriptional regulation of type I collagen synthesis.

Osteosclerosis and inhibition of human hematopoiesis in NOG mice expressing human Delta-like 1 in osteoblasts

NOD/Shi-scid IL2rγnull (NOG) mice with severe immunodeficiency are excellent recipients to generate "humanized" mice by the transplantation of human CD34(+) hematopoietic stem cells (HSCs). In this study, we developed NOG mice carrying a human Delta-like1 (DLL1) gene, which is a ligand of the Notch receptor and is known to be important in HSC maintenance and self-renewal. We also analyzed the effect of DLL1 signaling on human hematopoiesis and HSC maintenance using humanized DLL1 transgenic NOG mice. To develop DLL1 transgenic NOG (NOG-D1-Tg) mice, a transgenic vector consisting of a human DLL1 complementary DNA fragment placed downstream of the α1(I) collagen (Col1a1) promoter for expression specifically in osteoblasts was constructed. Human CD34(+) HSCs were transplanted into NOG-D1-Tg mice, and differentiation of lymphoid or myeloid lineage cells from human HSCs and maintenance of HSCs in bone marrow were analyzed. Severe osteosclerosis accompanied by increased bone mass and a decreased number of bone marrow cells were observed in NOG-D1-Tg mice. After human HSC transplantation, development of human B lymphocytes, but not T lymphocytes, was significantly suppressed in both bone marrow and the periphery of NOG-D1-Tg mice. Contrary to the initial expectation, retention of human CD34(+) HSCs was inhibited in the bone marrow of NOG-D1-Tg mice. In conclusion, our data suggest that the development of human B lymphocytes and HSC maintenance in osteosclerotic bone may be suppressed by introducing DLL1. These unique humanized mice with sclerotic bone reconstituted by human HSCs are useful models of hematopoiesis in patients with osteosclerosis, such as osteopetrosis, and for investigation of osteogenesis via Notch signaling.

Hypermethylation of collagen α2 (I) gene (COL1A2) is an independent predictor of survival in head and neck cancer

Collagen production plays a role in the development of tumors from cancer cells. The aim of the present study is to examine the involvement of epigenetic alteration of Collagen α2 (I) (COL1A2) gene expression in cases of head and neck squamous cell carcinoma (HNSCC). COL1A2 expression was examined in a panel of cell lines using RT-PCR. The methylation status of the COL1A2 promoter was studied using bisulfate sequencing and methylation-specific PCR (MSP). COL1A2 expression was absent in 6 of 11 (54.5%) UM-SCC cell lines, whereas three nonmalignant cell lines had stable expressions. MSP analysis showed that 46/98 (46.9%) contained methylated alleles. COL1A2 methylation was significantly correlated with tumor size (P = 0.041), lymph node status (P = 0.008), tumor stage (P = 0.011), H-cadherin methylation (P = 0.039) and disease-free survival (P = 0.005). On multivariate Cox proportional hazard regression, which included age, sex, smoking status, and alcohol exposure, both tumor stage and COL1A2 methylation remained independent prognostic factors. This study suggests that CpG hypermethylation is a likely mechanism of COL1A2 gene inactivation, supporting the hypothesis that the COL1A2 gene may play a role in the tumorigenesis of HNSCC and may serve as an important biomarker.