Biomedical subjects

Andre J Van Wijnen

Publications and source records attributed to Andre J Van Wijnen.

8 recordsLinked to original sources

PubMed2003

Osteoblast-related transcription factors Runx2 (Cbfa1/AML3) and MSX2 mediate the expression of bone sialoprotein in human metastatic breast cancer cells.

Human breast cancers are known to preferentially metastasize to skeletal sites, however, the mechanisms that mediate the skeletal preference (orthotropism) of specific types of cancers remains poorly understood. There is a significant clinical correlation between the expression of bone sialoprotein (BSP) and skeletal metastasis of breast cancers. Our laboratory, as well as others, have proposed the concept that skeletal selective metastasis and associated disease may be attributable to a mimicry of skeletal cellular phenotypes by metastasizing cancer cells. We hypothesize that breast cancer cell expression of phenotypic properties of skeletal cell types, including BSP as one component of that phenotype, is the result of ectopic expression or activity of one or more central transcriptional regulators of bone cell gene expression. To test this hypothesis, we examined the molecular mechanisms that regulate bsp expression in human breast cancer cell lines with previously characterized metastatic potentials. Our results demonstrate that the majority of the distal bsp promoter sequences act to repress BSP expression in cancer cells and that most of the promoter activity resides in the proximal -110 bp of the bsp promoter. In this region, we identified a putative Runx binding element providing a basis for a mechanism for skeletal gene activation. Our results demonstrate that Runx2 is ectopically expressed in breast cancer cells and that one isoform of Runx2 can activate bsp expression in these cells. In addition, we observe that bsp expression is additionally regulated by the homeodomain factor Msx2, another regulator of osteoblast-associated genes. Thus, this is the first report of osteoblast-related transcription factors being expressed in human breast cancer cells and provides a component of a mechanism that may explain the osteoblastic phenotype of human breast cancer cells that preferentially metastasize to bone.

Breast Neoplasms↗

PubMed2003

Inhibitory effects of insulin-like growth factor-1 and osteogenic protein-1 on fibronectin fragment- and interleukin-1beta-stimulated matrix metalloproteinase-13 expression in human chondrocytes.

Matrix metalloproteinase-13 (collagenase-3), a member of the family of matrix metalloproteinases (MMPs), plays a major pathological role in the cartilage destruction of arthritis. A dramatic up-regulation of MMP-13 by inflammatory cytokines such as interleukin (IL)-1beta or by fibronectin fragments has been observed in chondrocytes. In this study, we investigated the inhibitory effects of insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) on the expression of MMP-13, which was induced by fibronectin fragment or IL-1beta in human immortalized or human primary chondrocytes. IGF-1 and OP-1 each significantly reduced the basal level as well as fibronectin fragment- or IL-1beta-stimulated transcription of the MMP-13 gene in a dose-dependent fashion with the corresponding decreases in the protein level of MMP-13. The most prominent suppressive effect was observed by the combination of IGF-1 and OP-1, which decreased the basal promoter activity by 60% and almost completely abrogated the fibronectin fragment-stimulated MMP-13 promoter activity. OP-1 was found to enhance mRNA levels of IGF-1 and the IGF-1 receptor, the latter of which appeared to be responsible for the combined effect of IGF-1 and OP-1. The suppressive effect of IGF-1 and OP-1 on MMP-13 expression was due in part to down-regulation of the expression of pro-inflammatory cytokines and the activity of their intermediate molecules, including NF-kappaB and AP-1 factors. We propose that IGF-1 and OP-1 could be key physiological regulators of MMP-13 gene expression and that the combination of IGF-1 and OP-1 may be useful in controlling the excess catabolic activity in arthritis.

Blotting, Western↗

PubMed2003

Transcriptional induction of the osteocalcin gene during osteoblast differentiation involves acetylation of histones h3 and h4.

The remodeling of chromatin is required for tissue-specific gene activation to permit interactions of transcription factors and coregulators with their cognate elements. Here, we investigate the chromatin-mediated mechanisms by which the bone-specific osteocalcin (OC) gene is transcriptionally activated during cessation of cell growth in ROS 17/2.8 osteosarcoma cells and during normal osteoblast differentiation. Acetylation of histones H3 and H4 at the OC gene promoter was assayed during the proliferative and postproliferative stages of cell growth by using chromatin immunoprecipitation assays with antibodies that recognize different acetylated forms of histones H3 or H4. The results show that the promoter and coding regions of the OC gene contain very low levels of acetylated histones H3 and H4 during the proliferative period of osteoblast differentiation when the OC gene is inactive. Active expression of the OC gene in mature osteoblasts and confluent ROS 17/2.8 cells is functionally linked to preferential acetylation of histone H4 and, to a lesser extent, to acetylation of histone H3. Histone acetylation at the loci for RUNX2 (CBFA1), alkaline phosphatase, bone sialoprotein, osteopontin, and the cell growth regulator p21, which are expressed throughout osteoblast differentiation, is not altered postproliferatively. We conclude that acetylation of histones H3 and H4 is functionally coupled to the chromatin remodeling events that mediate the developmental induction of OC gene transcription in bone cells.

Acetylation↗

PubMed2003

Runx1/AML1 hematopoietic transcription factor contributes to skeletal development in vivo.

The requirement of Runx2 (Cbfal/AML3), a runt homology domain transcription factor essential for bone formation and osteoblast differentiation, is well established. Although Runx2 is expressed in the developing embryo prior to ossification, yet in the absence of Runx2 initial formation of the skeleton is normal, suggesting a potential redundancy in function of Runx family members. Here we addressed expression of the hematopoietic family member Runx1 (AML1/Cbfa2) in relation to skeletal development using a LacZ knock-in mouse model (Runx1(lz/+)). The resulting fusion protein reflects Runx1 promoter activity in its native context. Our studies show that Runx1 is expressed by prechondrocytic tissue forming the cartilaginous anlagen in the embryo, resting zone chondrocytes, suture lines of the calvarium, and in periosteal and perichondral membranes of all bone. Runx1 continues to be expressed in these tissues in adult mice, but is absent in mature cartilage or mineralized bone. However, hyaline cartilage outside the bone environment (trachea, xiphoid tissues), and epithelium of many soft tissues (trachea, thyroid, lung, skin) also express Runx1. The robust expression of Runx1 in vivo in chondroblasts at sites of cartilage growth and in osteoblasts at sites of new bone formation, suggests that Runx1 expression may be related to osteochondroprogenitor cell differentiation. This observation is further supported by high expression of Runx1 in ex vivo cultures of marrow stromal cells and calvarial derived osteoblasts from Runx1(lz/+) mice. These data indicate that Runx1 may contribute to the early stages of skeletogenesis and continues to function in the progenitor cells of tissues that support bone formation in the adult.

Animals↗

PubMed2003

Tumor suppressor pRB functions as a co-repressor of the CCAAT displacement protein (CDP/cut) to regulate cell cycle controlled histone H4 transcription.

The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key proliferation-related function as the DNA binding subunit of the cell cycle controlled HiNF-D complex. HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell-cycle dependent histone genes, which are transcriptionally and coordinately activated at the G(1)/S phase transition independent of E2F. The tumor suppressor pRB/p105 is an intrinsic component of the HiNF-D complex. However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined. Using transient transfections, we show that CDP represses the H4 gene promoter and that pRB functions with CDP as a co-repressor. Direct physical interaction between CDP and pRB was observed in glutathione-S-transferase (GST) pull-down assays. Furthermore, interactions between these proteins were established by yeast and mammalian two-hybrid experiments and co-immunoprecipitation assays. Confocal microscopy shows that subsets of each protein are co-localized in situ. Using a series of pRB mutants, we find that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB. Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters.

Animals↗

PubMed2003

Maintenance of open chromatin and selective genomic occupancy at the cell cycle-regulated histone H4 promoter during differentiation of HL-60 promyelocytic leukemia cells.

During the shutdown of proliferation and onset of differentiation of HL-60 promyelocytic leukemia cells, expression of the cell cycle-dependent histone genes is downregulated at the level of transcription. To address the mechanism by which this regulation occurs, we examined the chromatin structure of the histone H4/n (FO108, H4FN) gene locus. Micrococcal nuclease, DNase I, and restriction enzymes show similar cleavage sites and levels of sensitivity at the H4/n locus in both proliferating and differentiated HL-60 cells. In contrast, differentiation-related activation of the cyclin-dependent kinase inhibitor p21(cip1/WAF1) gene is accompanied by increased nuclease hypersensitivity. Chromatin immunoprecipitation assays of the H4/n gene reveal that acetylated histones H3 and H4 are maintained at the same levels in proliferating and postproliferative cells. Thus, the chromatin of the H4/n locus remains in an open state even after transcription ceases. Using ligation-mediated PCR to visualize genomic DNase I footprints at single-nucleotide resolution, we find that protein occupancy at the site II cell cycle element is selectively diminished in differentiated cells while the site I element remains occupied. Decreased occupancy of site II is reflected by loss of the site II binding protein HiNF-P. We conclude that H4 gene transcription during differentiation is downregulated by modulating protein interaction at the site II cell cycle element and that retention of an open chromatin conformation may be associated with site I occupancy.

Acetylation↗

PubMed2003

Regulation of the bone-specific osteocalcin gene by p300 requires Runx2/Cbfa1 and the vitamin D3 receptor but not p300 intrinsic histone acetyltransferase activity.

p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors. p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation. In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D(3) via the vitamin D(3) receptor complex. Therefore, we addressed p300 control of basal and vitamin D(3)-enhanced activity of the OC promoter. We find that transient overexpression of p300 results in a significant dose-dependent increase of both basal and vitamin D(3)-stimulated OC gene activity. This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element. In addition, by coimmunoprecipitation, we show that the endogenous Runx2/Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations. We also demonstrate by chromatin immunoprecipitation assays that p300, Runx2/Cbfa1, and 1alpha,25-dihydroxyvitamin D(3) receptor interact with the OC promoter in intact osteoblastic cells expressing this gene. The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, as a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300. On the basis of these results, we propose that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D(3).

Acetyltransferases↗

PubMed2002

Histone acetylation in vivo at the osteocalcin locus is functionally linked to vitamin D-dependent, bone tissue-specific transcription.

The accessibility of regulatory elements in chromatin represents a principal rate-limiting parameter of gene transcription and is modulated by enzymatic transcriptional co-factors that alter the topology of chromatin or covalently modify histones (e.g. by acetylation). The bone-specific activation and 1,25-dihydroxyvitamin D(3) enhancement of osteocalcin (OC) gene transcription are both functionally linked to modifications in nucleosomal organization. The initiation of tissue-specific basal transcription is accompanied by the induction of two DNase I hypersensitive sites, and this chromatin remodeling event requires binding of the key osteogenic factor RUNX2/CBFA1 to the OC promoter. Here, we analyzed the acetylation status of histones H3 and H4 when the OC gene is active (in osteoblastic ROS17/2.8 cells) or inactive (in fibroblastic ROS24/1 cells) using chromatin immunoprecipitation assays. We find that acetylated histone H3 and H4 proteins are associated with the OC promoter only when the gene is transcriptionally active and that the acetylation status is relatively uniform across the OC locus under basal conditions. Acetylation of H4 at the OC gene is selectively increased following vitamin D(3) enhancement of OC transcription, with the most prominent changes occurring in the region between the vitamin D(3) enhancer and basal promoter. Thus, our results suggest functional linkage of H3 and H4 acetylation in specific regions of the OC promoter to chromatin remodeling that accompanies tissue-specific transcriptional activation and vitamin D enhancement of OC gene expression. These findings provide mechanistic insights into bone-specific gene activation within a native genomic context in response to steroid hormone-related regulatory cues.

Acetylation↗