COL1A2 Promoter Research Articles

Abstract A43: Lysyl oxidases are poor therapeutic targets in pancreatic cancer

Abstract A characteristic feature of pancreatic cancer is the predominant stroma, which consists of fibroblasts, immune cells and a significant deposition of extracellular matrix (ECM). The stroma accounts for over eighty percent of the pancreatic tumor mass and plays significant, yet poorly understood, roles in tumor progression and therapeutic response. One major component of the ECM in pancreatic cancer is fibrillar type I collagen, which has been shown to serve as a barrier to chemotherapy and to facilitate metastatic dissemination of breast cancer cells through integrin-mediated signaling. The cross-linking of type I collagen mediated by the lysyl oxidases (LOX) is elevated in invasive cancers and inversely correlates with metastatic free survival in breast cancer patients. Moreover, inhibition of LOX enzymes in mouse models of breast cancer decreases tumor size, latency, and metastasis. Given the abundance of type I collagen and the high expression of several LOX family members in pancreatic cancer, we examined whether inhibiting these enzymes could limit the progression of pancreatic cancer in mouse models. Using syngeneic, orthotopic transplantation models of pancreatic cancer - with significant fibrosis - we determined the effects of lysyl oxidases on pancreatic cancer. First, we established that both LOX and LOX-like 2 (LOXL2) were expressed much higher in tumors than in normal pancreatic tissue. However, inhibition of LOX activity, using the small molecule inhibitor β-aminoproprionitirile, significantly increased tumor wet weight and metastasis to the liver and spleen – the exact opposite results of those reported in breast cancer mouse models. Similarly, silencing the expression of LOXL2 in cancer cells, using RNA interference, also resulted in increased tumor growth. To examine why LOX inhibition has this surprisingly negative effect on pancreatic cancer progression, we tested the effects of LOX enzyme inhibition on collagen structure by picrosirius red stains, which visualize fibrillar collagens when tissues are observed under cross-polarized light. LOX inhibition promoted the deposition of fibrillar collagen in the tumor. To determine how this altered collagen deposition influenced cell behavior, we performed confocal microscope imaging in live tumor-bearing mice that express a fusion protein in which GFPtopaz is inserted into the N-terminus of α2(I)- collagen and expression is driven by the 3.6kb Col1a1 promoter [α2(I)-col-GFP mice]. Live imaging of red fluorescent protein expressing cancer cells in pancreatic tumors in the α2(I)-col-GFP mice showed a rich deposit of collagen and migration of cancer cells on linear collagen. Imaging confirmed that administration of the LOX inhibitor for just 3 days resulted in thicker collagen fibers than the control treated tumors. We are currently examining the underlying mechanisms responsible for the opposite responses of LOX targeting in breast and pancreatic cancer. In conclusion, our data strongly suggest that targeting collagen cross-linking enzymes would be a poor strategy for the treatment of pancreatic cancer. Citation Format: Mario A. Shields, Pascal Maguin, Priyanka Kumar, Sarah L. Dallas, Mikala Egeblad. Lysyl oxidases are poor therapeutic targets in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A43.

Temporo-spatial requirement of Smad4 in dentin formation

The TGF-β/BMP family plays an important role in multiple stages of tooth development. TGF-β/BMP signaling is required for odontoblast differentiation and dentin formation; however, the precise molecular mechanisms underlying dentin formation remain unclear. To address the role of TGF-β/BMP signaling in dentin formation, we analyzed mice in which Smad4, a key intracellular mediator of TGF-β/BMP signaling, was subjected to tissue-specific ablation under the control of Dspp, OC, or Col1a1 promoters. Three independent Smad4 conditional knockout mice exhibited various dentin defects in the crowns and roots of their molars depending on the transactivator. In all mutant molars, crown dentin thickness was thinner than that of the control. In addition, impaired dentin was found in the cervical region and root furcation area. Although the initial differentiation of odontoblasts was normal, odontoblast polarity abruptly decreased and the expression of Col1a1, OC, and Dspp was reduced in the odontoblasts of mutant molars. In Dspp-Cre-mediated Smad4 disruption mice, primary dentin formation was slightly delayed, while secondary dentin formation was severely affected in the cervical region of the molars. These results indicate that TGF-β/BMP signaling is required for odontoblast maturation and dentin formation in a stage- and site-dependent manner.

Microtubule-associated protein tau (Mapt) is expressed in terminally differentiated odontoblasts and severely down-regulated in morphologically disturbed odontoblasts of Runx2 transgenic mice

Runx2 is an essential transcription factor for osteoblast and odontoblast differentiation and the terminal differentiation of chondrocytes. We have previously shown that the terminal differentiation of odontoblasts is inhibited in Runx2 transgenic {Tg(Col1a1-Runx2)} mice under the control of the 2.3-kb Col1a1 promoter, which directs the transgene expression to osteoblasts and odontoblasts. Odontoblasts show severe reductions in Dspp and nestin expression and lose their characteristic polarized morphology, including a long process extending to dentin, in Tg(Col1a1-Runx2) mice. We study the molecular mechanism of odontoblast morphogenesis by comparing gene expression in the molars of wild-type and Tg(Col1a1-Runx2) mice, focusing on cytoskeleton-related genes. Using microarray, we found that the gene expression of microtubule-associated protein tau (Mapt), a neuronal phosphoprotein with important roles in neuronal biology and microtubule dynamics and assembly, was high in wild-type molars but severely reduced in Tg(Col1a1-Runx2) molars. Immunohistochemical analysis revealed that Mapt was specifically expressed in terminally differentiated odontoblasts including their processes in wild-type molars but its expression was barely detectable in Tg(Col1a1-Runx2) molars. Double-staining of Mapt and Runx2 showed their reciprocal expression in odontoblasts. Mapt and tubulin co-localized in odontoblasts in wild-type molars. Immunoelectron microscopic analysis demonstrated Mapt lying around α-tubulin-positive filamentous structures in odontoblast processes. Thus, Mapt is a useful marker for terminally differentiated odontoblasts and might play an important role in odontoblast morphogenesis.

Osteoblast Menin Regulates Bone Mass in Vivo

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1(f/f)) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1(f/f) mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.

A novel role of annexin A2 in human type I collagen gene expression.

ABSTRACTThe fibrillar collagen scaffold of the extracellular matrix provides a structural framework for cells in tissues and regulates intercellular communication; its disregulation has been associated with tumour development and progression. Previous work has shown that expression of type I collagen, the most abundant mammalian extracellular matrix protein, is decreased in chemically or virally transformed cells. This negative regulation could be mapped to a proximal COL1A2 promoter element spanning a CME (Collagen Modulating Element) site in SV40‐transformed human fibroblasts (SV‐WI38) that binds an unknown repressing protein. By magnetic bead pull‐down, we observed a multi‐protein complex bound to the CME with preference for single‐stranded over conventional double‐stranded DNA. MALDI‐TOF mass spectrometry of the CME‐binding protein complex revealed involvement of nuclear annexin A2 (AnxA2) which was increased in SV40‐transformed cells. Further EMSA analysis demonstrated that AnxA2 did not directly bind to the DNA but stabilised the complex and led to an increase in protein binding to the CME in SV‐WI38 but not untransformed WI38 cells. Knockdown of AnxA2 by siRNA increased type I collagen production in both WI38 and SV‐WI38 cells; however, these effects were not mediated at the transcriptional level. Rather, our data indicate a novel functional role of AnxA2 in the negative post‐transcriptional regulation of type I collagen synthesis in human fibroblasts. In SV40‐transformed cells, AnxA2 is accumulated at the proximal COL1A2 promoter region, suggesting close association with the transcriptional machinery that possibly facilitates binding to the emerging mRNA, eventually contributing to overall repression of type I collagen protein synthesis. J. Cell. Biochem. 116: 408–417, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

The Anti-Osteoanabolic Function of Sclerostin Is Blunted in Mice Carrying a High Bone Mass Mutation of Lrp5.

Activating mutations of the putative Wnt co-receptor Lrp5 or inactivating mutations of the secreted molecule Sclerostin cause excessive bone formation in mice and humans. Previous studies have suggested that Sclerostin functions as an Lrp5 antagonist, yet clear in vivo evidence was still missing, and alternative mechanisms have been discussed. Moreover, because osteoblast-specific inactivation of β-catenin, the major intracellular mediator of canonical Wnt signaling, primarily affected bone resorption, it remained questionable, whether Sclerostin truly acts as a Wnt signaling antagonist by interacting with Lrp5. In an attempt to address this relevant question, we generated a mouse model (Col1a1-Sost) with transgenic overexpression of Sclerostin under the control of a 2.3-kb Col1a1 promoter fragment. These mice displayed the expected low bone mass phenotype as a consequence of reduced bone formation. The Col1a1-Sost mice were then crossed with two mouse lines carrying different high bone mass mutations of Lrp5 (Lrp5(A170V) and Lrp5(G213V)), both of them potentially interfering with Sclerostin binding. Using µCT-scanning and histomorphometry we found that the anti-osteoanabolic influence of Sclerostin overexpression was not observed in Lrp5(A213V/A213V) mice and strongly reduced in Lrp5(A170V/A170V) mice. As a control we applied the same strategy with mice overexpressing the transmembrane Wnt signaling antagonist Krm2 and found that the anti-osteoanabolic influence of the Col1a1-Krm2 transgene was not affected by either of the Lrp5 mutations. Taken together, our data support the concept that Sclerostin inhibits bone formation through Lrp5 interaction, yet their physiological relevance remains to be established.

A TRANSGENIC APPROACH TO STUDY THE EFFECT OF CARDIAC FIBROBLAST-SPECIFIC ABLATION OF IL-1 SIGNALLING ON MYOCARDIAL REMODELLING

<h3>Introduction</h3> Cardiac fibroblasts (CF) play a critical role in the repair of the heart following MI, but aberrant remodelling can lead to fibrosis and heart failure. We have previously demonstrated that interleukin-1 (IL-1), a key proinflammatory cytokine released following MI, is a potent modulator of CF function. The aim of this study was to develop a mouse model with fibroblast-specific ablation of IL-1 signalling through deletion of Myd88. <h3>Methods</h3> We generated a mouse line with tamoxifen-inducible Cre-recombinase under control of the fibroblast-specific Col1a2 promoter, and loxP sites flanking exon 3 of the Myd88 gene, an essential component of the IL-1 receptor signalling complex. Mice were administered tamoxifen (75–100 mg/kg; i.p; 5 days) to induce Myd88 deletion. DNA was obtained from ear notches and RNA from cultured CF. Myd88 DNA deletion efficiency and mRNA expression levels were determined by real-time RT-PCR. All data are expressed as mean±SEM %GAPDH. <h3>Results</h3> Tamoxifen induced a 32% reduction in Myd88 DNA (Cre- 0.172±0.017 (n=7) vs. Cre+ 0.117±0.015 (n=9); P=0.049) and a 63% reduction in CF Myd88 mRNA expression (Cre- 1.64±0.27 (n=4) vs. Cre+ 0.60±0.25 (n=4); P=0.023). Expression of DDR2 mRNA, a fibroblast marker, was unaffected (Cre- 0.91±0.06 vs. Cre+ 0.77±0.10; P=0.279) indicating the specificity of Myd88 deletion. Increasing tamoxifen dose (100 mg/kg) did not enhance deletion efficiency. <h3>Conclusions</h3> We have successfully reduced expression of Myd88 in CF from tamoxifen-treated Cre+ animals. To obtain greater deletion efficiency we propose using a PGK-Cre line to globally delete one Myd88 allele in the germline prior to tamoxifen-induced ablation in CF.

Core binding factor β of osteoblasts maintains cortical bone mass via stabilization of Runx2 in mice.

Core binding factor beta (Cbfβ), the partner protein of Runx family transcription factors, enhances Runx function by increasing the binding of Runx to DNA. Null mutations of Cbfb result in embryonic death, which can be rescued by restoring fetal hematopoiesis but only until birth, where bone formation is still nearly absent. Here, we address a direct role of Cbfβ in skeletal homeostasis by generating osteoblast-specific Cbfβ-deficient mice (Cbfb(Δob/Δob) ) from Cbfb-floxed mice crossed with mice expressing Cre from the Col1a1 promoter. Cbfb(Δob/Δob) mice showed normal growth and development but exhibited reduced bone mass, particularly of cortical bone. The reduction of bone mass in Cbfb(Δob/Δob) mice is similar to the phenotype of mice with haploinsufficiency of Runx2. Although the number of osteoblasts remained unchanged, the number of active osteoblasts decreased in Cbfb(Δob/Δob) mice and resulted in lower mineral apposition rate. Immunohistochemical and quantitative real-time PCR analyses showed that the expression of osteogenic markers, including Runx2, osterix, osteocalcin, and osteopontin, was significantly repressed in Cbfb(Δob/Δob) mice compared with wild-type mice. Cbfβ deficiency also reduced Runx2 protein levels in osteoblasts. The mechanism was revealed by forced expression of Cbfβ, which increased Runx2 protein levels in vitro by inhibiting polyubiquitination-mediated proteosomal degradation. Collectively, these findings indicate that Cbfβ stabilizes Runx2 in osteoblasts by forming a complex and thus facilitates the proper maintenance of bone mass, particularly cortical bone.

Role of Flightless-I (Drosophila) homolog in the transcription activation of type I collagen gene mediated by transforming growth factor beta

Flightless-I (Drosophila) homolog (FLII) is a nuclear receptor coactivator that is known to interact with other transcriptional regulators such as the SWI/SNF complex, an ATP-dependent chromatin-remodeling complex, at the promoter or enhancer region of estrogen receptor (ER)-α target genes. However, little is known about the role of FLII during transcription initiation in the transforming growth factor beta (TGFβ)/SMAD-dependent signaling pathway. Here, we demonstrate that FLII functions as a coactivator in the expression of type I collagen gene induced by TGFβ in A549 cells. FLII activates the reporter gene driven by COL1A2 promoter in a dose-dependent manner. Co-expression of GRIP1, CARM1, or p300 did not show any synergistic activation of transcription. Furthermore, the level of COL1A2 expression correlated with the endogenous level of FLII mRNA level. Depletion of FLII resulted in a reduction of TGFβ-induced expression of COL1A2 gene. In contrast, over-expression of FLII caused an increase in the endogenous expression of COL1A2. We also showed that FLII is associated with Brahma-related gene 1 (BRG1) as well as SMAD in A549 cells. Notably, the recruitment of BRG1 to the COL1A2 promoter region was decreased in FLII-depleted A549 cells, suggesting that FLII is required for TGFβ-induced chromatin remodeling, which is carried out by the SWI/SNF complex. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments revealed that depletion of FLII caused a reduction in chromatin accessibility at the COL1A2 promoter. These results suggest that FLII plays a critical role in TGFβ/SMAD-mediated transcription of the COL1A2 gene through its role in recruiting the SWI/SNF complex to facilitate chromatin accessibility.

Dkk‐1–Mediated Inhibition of Wnt Signaling in Bone Ameliorates Osteoarthritis in Mice

Wnt signaling is a master regulator of joint homeostasis, but its role in osteoarthritis (OA) remains unclear. This study was undertaken to characterize the activation of Wnt/β-catenin in knee joints of mice with OA and to assess how inhibiting this pathway in bone could affect cartilage. OA was induced by partial meniscectomy in Topgal mice and in transgenic mice overexpressing Dkk-1 under the control of the 2.3-kb Col1a1 promoter (Col1a1-Dkk-1-Tg mice). Wnt/β-catenin activation was assessed by X-Gal staining at baseline and at weeks 4, 6, and 9. Cartilage and bone damage was analyzed in Col1a1-Dkk-1-Tg mice with OA at week 6. Primary chondrocytes and cartilage explants were used to assess the effect of Dkk-1 on cartilage catabolism. In meniscectomized Topgal mice, Wnt was mainly activated in osteocytes from the subchondral bone at week 6 after OA induction, as well as in osteophytes and synovium at week 4. Chondrocytes from damaged zones expressed X-Gal from week 4. Dkk-1 expression was high in chondrocytes in control mouse knees (mean ± SEM 84.2 ± 3.1%) but decreased greatly in knees of meniscectomized mice from week 4 (mean ± SEM 14.4 ± 3.8%). The OA score was lower in meniscectomized Col1a1-Dkk-1-Tg mice at week 6 compared with wild-type mice (5.1 ± 0.6 versus 8.4 ± 0.6; P = 0.002). Subchondral bone fraction and osteophyte volume were decreased. However, cartilage explants from Col1a1-Dkk-1-Tg mice showed proteoglycan loss and increased NITEGE expression. Expression of vascular endothelial growth factor (VEGF) was reduced in osteoblasts from Col1a1-Dkk-1-Tg mice, thereby decreasing expression of messenger RNA for matrix metalloproteinases in chondrocytes. Wnt activation in OA affects the whole joint, particularly bone. Selective inhibition of this pathway in bone by Dkk-1 decreased OA severity through VEGF inhibition.

Aggrecan is required for growth plate cytoarchitecture and differentiation

The proteoglycan aggrecan is a prominent component of the extracellular matrix in growth plate cartilage. A naturally occurring, recessive, perinatally lethal mutation in the aggrecan core protein gene, cmdbc (Acancmd-Bc), that deletes the entire protein-coding sequence provided a model in which to characterize the phenotypic and morphologic effects of aggrecan deletion on skeletal development. We also generated a novel transgenic mouse, Tg(COL2A1-ACAN), that has the chick ACAN coding sequence driven by the mouse COL2A1 promoter to enable the production of cmdbc/cmdbc; Tg(COL2A1-ACAN) rescue embryos. These were used to assess the impact of aggrecan on growth plate organization, chondrocyte survival and proliferation, and the expression of mRNAs encoding chondrocyte differentiation markers and growth factors. Homozygous mutant (cmdbc/cmdbc) embryos exhibited severe defects in all skeletal elements with deformed and shortened (50%) limb elements. Expression of aggrecan in rescue embryos reversed the skeletal defects to varying degrees with a 20% increase in limb element length and near-full reversal (80%) of size and diameter of the ribcage and vertebrae. Aggrecan-null growth plates were devoid of matrix and lacked chondrocyte organization and differentiation, while those of the rescue embryos exhibited matrix production concomitant with partial zonation of chondrocytes having proliferative and hypertrophic morphologies. Deformation of the trachea, likely the cause of the mutation׳s lethality, was reduced in the rescue embryos. Aggrecan-null embryos also had abnormal patterns of COL10A1, SOX9, IHH, PTCH1, and FGFR3 mRNA expression in the growth plate. Expression of chick aggrecan in the rescue embryos notably increased COLX expression, accompanied by the reappearance of a hypertrophic zone and IHH expression. Significantly, in transgenic rescue embryos, the cell death and decreased proliferation phenotypes exhibited by the mutants were reversed; both were restored to wild-type levels. These findings suggest that aggrecan has a major role in regulating the expression of key growth factors and signaling molecules during development of cartilaginous tissue and is essential for proper chondrocyte organization, morphology, and survival during embryonic limb development.

Construction and validation of a dual-luciferase reporter gene system for screening and evaluating anti-liver fibrosis drugs that inhibit transcription of the gene encoding collagen I, chain a1

To construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain at1 (COL1A1). The full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter.All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing.Either the pcDNA3.1-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter gene system.This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1.Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system. The two TGFbeta1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed.The results of a dual-reporter gene assay showed that TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 co-expression the activity was below 2-fold (t =3.396, P =0.0274).The transcriptionactivated dual-reporter gene system was successfully established.The model drug, dexamethasone, effectively inhibited the activity of the COL 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035). The dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.

Cbfb Regulates Bone Development by Stabilizing Runx Family Proteins

Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA-binding capacity. Cbfb-deficient (Cbfb(-/-) ) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb(-/-) mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfb(fl/fl) mice with Dermo1 Cre knock-in mice. Cbfb(fl/fl/Cre) mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfb(fl/fl/Cre) chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfb(fl/fl/Cre) embryos compared with the respective protein in the respective tissue of Cbfb(fl/fl) embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfb(fl/fl/Cre) primary osteoblasts, and Runx2 protein was less stable in Cbfb(fl/fl/Cre) osteoblasts than Cbfb(fl/fl) osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons.

Sp7/Osterix induces the mouse pro-α2(I) collagen gene (Col1a2) expression via the proximal promoter in osteoblastic cells

Bone is essentially composed of two components, hydroxyapatite and extracellular matrix proteins. The extracellular matrix of bone is primary composed of collagen, mostly type I collagen, with lesser amounts of other types of collagen such as type V collagen. Osteoblast differentiation is a multi-step process in which many classes of factors function in a coordinated manner. Sp7/Osterix, which binds to G/C-rich sequences, is a transcription factor that contributes to osteoblast differentiation. The present study aimed to clarify the involvement of Sp7/Osterix with the proximal promoter region of the mouse Col1a2 gene containing multiple G/C-rich sequences exist. Consequently, a functional analysis of the proximal mouse Col1a2 promoter showed that a substitution mutation of the second G/C-rich sequence from the transcription site specifically decreased the activity of osteoblastic cells. In addition, the experiments of overexpression of Sp7/Osterix and treatment with its specific siRNA showed that this G/C-rich sequence is responsible for the specific expression in osteoblastic cells. Consistent with these data, Sp7/Osterix bound to the region and increased the expression of the Col1a2 gene in association with osteoblast differentiation in the culture system.

Key role of suppressor of cytokine signaling 3 in regulating gp130 cytokine-induced signaling and limiting chondrocyte responses during murine inflammatory arthritis.

To examine the impact of the gp130 cytokine family on murine articular cartilage and to explore a potential regulatory role of suppressor of cytokine signaling 3 (SOCS-3) in murine chondrocytes. In wild-type (WT) mouse chondrocytes, baseline receptor expression levels and gp130 cytokine-induced JAK/STAT signaling were determined by flow cytometry, and expression of SOCS-3 was assessed by quantitative polymerase chain reaction. The role of endogenous SOCS-3 was examined in cartilage explants and chondrocytes from mice with conditional deletion of Socs3 driven by the Col2a1 promoter in vitro (Socs3(Δ/Δcol2) ) and from mice during CD4+ T cell-dependent inflammatory monarthritis. Bone erosions in the murine joints were analyzed by micro-computed tomography. On chondrocytes from WT mice, gp130 and the oncostatin M (OSM) receptor were strongly expressed, whereas the transmembrane interleukin-6 (IL-6) receptor was expressed at much lower levels. Compared to other gp130 cytokines, OSM was the most potent activator of the JAK/STAT pathway and of SOCS-3 induction. Treatment of Socs3(Δ/Δcol2) mouse cartilage explants and chondrocytes with gp130 cytokines prolonged JAK/STAT signaling, enhanced cartilage degradation, increased the expression of Adamts4, Adamts5, and RANKL, and elevated the production of IL-6, granulocyte colony-stimulating factor, CXCL1, and CCL2. Socs3(Δ/Δcol2) mice developed exacerbated inflammation and joint damage in response to gp130 cytokine injections, and these histopathologic features were also observed in mice with inflammatory monarthritis. The results of this study highlight a key role for SOCS-3 in regulating chondrocyte responses during inflammatory arthritis. Within the gp130 cytokine family, OSM is a potent stimulus of chondrocyte responses, while IL-6 probably signals via trans-signaling. The gp130 cytokine-driven production of RANKL in chondrocytes may link chondrocyte activation and bone remodeling during inflammatory arthritis. Thus, these findings suggest that the inhibition of OSM might reduce the development and severity of structural joint damage during inflammatory arthritis.

Analyses on the mechanisms that underlie the chondroprotective properties of calcitonin

IntroductionCalcitonin (CT) has recently been shown to display chondroprotective effects. Here, we investigate the putative mechanisms by which CT delivers these actions. MethodsImmortalized C-28/I2 cells or primary adult human articular chondrocytes (AHAC) were cultured in high-density micromasses to investigate: (i) CT anabolic effects using qPCR and immuhistochemistry analysis; (ii) CT anti-apoptotic effects using quantitation of Bax/Bcl gene products ratio, TUNEL assay and caspase-3 expression; (iii) CT effects on CREB, COL2A1 and NFAT transcription factors. ResultsCT (10−10–10−8nM) induced significant up-regulation of cartilage phenotypic markers (SOX9, COL2A1 and ACAN), with down-regulation of catabolic (MMP1 and MMP13 and ADAMTS5) gene products both in resting and inflammatory conditions. This was mirrored by an augmented production of type II collagen and accumulation of glycosaminoglycan- and proteoglycan-rich extracellular matrix in vitro. Mechanistic analyses revealed only partial involvement of cyclic AMP formation in these effects of CT. Congruently, using reporter assays for specific transcription factors, there was no indication for CREB activation, whereas the COL2A1 promoter was genuinely and directly activated by cell exposure to CT. Phenotypically, these mechanisms supported the ability of CT, whilst inactive on its own, to counteract the pro-apoptotic effects of IL-1β, demonstrated by TUNEL-positive staining of chondrocytes and ratio of BAX/BCL genes products. ConclusionThese data may provide a novel lead for the development of CT-based chondroprotective strategies that rely on the engagement of mechanisms that lead to augmented chondrocyte anabolism and inhibited chondrocyte apoptosis.

Notch signaling regulates col1α1 and col1α2 expression in airway fibroblasts.

Subepithelial fibrosis is one of the common pathological features of asthmatic airway remodeling. During subepithelial fibrosis, type I collagen becomes the most abundant extracellular protein component. Studies have shown that Notch signaling participates in the progression of fibrosis; however, whether Notch signaling is involved in regulating type I collagen expression in airway fibroblasts remains unclear. The aim of the present study was to examine whether Notch signaling can regulate type I collagen expression in airway fibroblasts and to explore the underlying molecular mechanisms. Here, the expression of Notch signaling components was examined in mouse L929 cells and human MRC-5 cells. After upregulating or downregulating Notch signaling in these cell lines, col1α1 and col1α2 expression was examined. Using gene reporter assays, site-directed mutagenesis, and ChIP assays, the role of Hes1 binding sites in both the mouse and human COL1A1 and COL1A2 promoters was investigated. This study revealed that Notch signaling-related molecules (including Notch1, Hes1, and others) are expressed in L929 and MRC-5 cells and that Notch signaling regulates the expression of col1α1 and col1α2 in both cell lines. Additionally, over-expression of the Notch intracellular domain resulted in activation of the COL1A1 and COL1A2 promoters, and site-directed mutagenesis reporter assays revealed that Hes1 proteins might augment both mouse and human COL1A1 and COL1A2 promoter activity. Furthermore, ChIP assays confirmed that Hes1 binds to the COL1A1 and COL1A2 promoters in both L929 and MRC-5 cells. Therefore, it is reasonable to assume that Notch signaling can directly upregulate COL1A1 and COL1A2 promoter activity through a Hes1-dependent mechanism, which could serve as a possible target for pharmacotherapy of airway subepithelial fibrosis.

Up-regulation of type II collagen gene by 17β-estradiol in articular chondrocytes involves Sp1/3, Sox-9, and estrogen receptor α.

The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17β-estradiol (17β-E2) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17β-E2 stimulates, via its receptor human estrogen receptor α 66 (hERα66), type II collagen expression in differentiated and dedifferentiated (reflecting the OA phenotype) articular chondrocytes. Transactivation of type II collagen gene (COL2A1) by ligand-independent transactivation domain (AF-1) of hERα66 was mediated by "GC" binding sites of the -266/-63-bp promoter, through physical interactions between ERα, Sp1/Sp3, Sox9, and p300, as demonstrated in chromatin immunoprecipitation (ChIP) and Re-Chromatin Immuno-Precipitation (Re-ChIP) assays in primary and dedifferentiated cells. 17β-E2 and hERα66 increased the DNA-binding activities of Sp1/Sp3 and Sox-9 to both COL2A1 promoter and enhancer regions. Besides, Sp1, Sp3, and Sox-9 small interfering RNAs (siRNAs) prevented hERα66-induced transactivation of COL2A1, suggesting that these factors and their respective cis-regions are required for hERα66-mediated COL2A1 up-regulation. Our results highlight the genomic pathway by which 17β-E2 and hERα66 modulate Sp1/Sp3 heteromer binding activity and simultaneously participate in the recruitment of the essential factors Sox-9 and p300 involved respectively in the chondrocyte-differentiated status and COL2A1 transcriptional activation. These novel findings could therefore be attractive for tissue engineering of cartilage in OA, by the fact that 17β-E2 could promote chondrocyte redifferentiation. 17β-E2 up-regulates type II collagen gene expression in articular chondrocytes. An ERα66/Sp1/Sp3/Sox-9/p300 protein complex mediates this stimulatory effect. This heteromeric complex interacts and binds to Col2a1 promoter and enhancer in vivo. Our findings highlight a new regulatory mechanism for 17β-E2 action in chondrocytes. 17β-E2 might be an attractive candidate for cartilage engineering applications.

Indian hedgehog signaling regulates transcription and expression of collagen type X via Runx2/Smads interactions.

Indian hedgehog (Ihh) is essential for chondrocyte differentiation and endochondral ossification and acts with parathyroid hormone-related peptide in a negative feedback loop to regulate early chondrocyte differentiation and entry to hypertrophic differentiation. Independent of this function, we and others recently reported independent Ihh functions to promote chondrocyte hypertrophy and matrix mineralization in vivo and in vitro. However, the molecular mechanisms for these actions and their functional significance are still unknown. We recently discovered that Ihh overexpression in chondrocytes stimulated the expression of late chondrocyte differentiation markers and induced matrix mineralization. Focusing on collagen type X (Col10α1) expression and transcription, we observed that hedgehog downstream transcription factors GLI-Krüppel family members (Gli) 1/2 increased COL10A1 promoter activity and identified a novel Gli1/2 response element in the 250-bp basic promoter. In addition, we found that Ihh induced Runx2 expression in chondrocytes without up-regulating other modulators of chondrocyte maturation such as Mef2c, Foxa2, and Foxa3. Runx2 promoted Col10α1 expression in cooperation with Ihh. Further analyses using promoter assays, immunofluorescence, and binding assays showed the interaction of Gli1/2 in a complex with Runx2/Smads induces chondrocyte differentiation. Finally, we could demonstrate that Ihh promotes in vitro matrix mineralization using similar molecular mechanisms. Our data provide an in vitro mechanism for Ihh signaling to positively regulate Col10α1 transcription. Thus, Ihh signaling could be an important player for not only early chondrocyte differentiation but maturation and calcification of chondrocytes.

Shell extracts from the marine bivalve Pecten maximus regulate the synthesis of extracellular matrix in primary cultured human skin fibroblasts.

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the −112/−61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications.