A. J. Van Wijnen

Regulatory roles of Runx2 in metastatic tumor and cancer cell interactions with bone

The three mammalian Runt homology domain transcription factors (Runx1, Runx2, Runx3) support biological control by functioning as master regulatory genes for the differentiation of distinct tissues. Runx proteins also function as cell context-dependent tumor suppressors or oncogenes. Abnormalities in Runx mediated gene expression are linked to cell transformation and tumor progression. Runx2 is expressed in mesenchymal linage cells committed to the osteoblast phenotype and is essential for bone formation. This skeletal transcription factor is aberrantly expressed at high levels in breast and prostate tumors and cells that aggressively metastasize to the bone environment. In cancer cells, Runx2 activates expression of bone matrix and adhesion proteins, matrix metalloproteinases and angiogenic factors that have long been associated with metastasis. In addition, Runx2 mediates the responses of cells to signaling pathways hyperactive in tumors, including BMP/TGFβ and other growth factor signals. Runx2 forms co-regulatory complexes with Smads and other co-activator and co-repressor proteins that are organized in subnuclear domains to regulate gene transcription. These activities of Runx2 contribute to tumor growth in bone and the accompanying osteolytic disease, established by interfering with Runx2 functions in metastatic breast cancer cells. Inhibition of Runx2 in MDA-MB-231 cells transplanted to bone decreased tumorigenesis and prevented osteolysis. This review evaluates evidence that Runx2 regulates early metastatic events in breast and prostate cancers, tumor growth, and osteolytic bone disease. Consideration is given to the potential for inhibition of this transcription factor as a therapeutic strategy upstream of the regulatory events contributing to the complexity of metastasis to bone.

Runx2 association with progression of prostate cancer in patients: mechanisms mediating bone osteolysis and osteoblastic metastatic lesions.

Runx2, a bone-specific transcriptional regulator, is abnormally expressed in highly metastatic prostate cancer cells. Here, we identified the functional activities of Runx2 in facilitating tumor growth and osteolysis. Our studies show that negligible Runx2 is found in normal prostate epithelial and non-metastatic LNCaP prostate cancer cells. In the intra-tibial metastasis model, high Runx2 levels are associated with development of large tumors, increased expression of metastasis-related genes (MMP9, MMP13, VEGF, Osteopontin) and secreted bone-resorbing factors (PTHrP, IL8) promoting osteolytic disease. Runx2 siRNA treatment of PC3 cells decreased cell migration and invasion through Matrigel in vitro, and in vivo shRunx2 expression in PC3 cells blocked their ability to survive in the bone microenvironment. Mechanisms of Runx2 function were identified in co-culture studies showing that PC3 cells promote osteoclastogenesis and inhibit osteoblast activity. The clinical significance of these findings is supported by human tissue microarray studies of prostate tumors at stages of cancer progression, in which Runx2 is expressed in both adenocarcinomas and metastatic tumors. Together these findings indicate that Runx2 is a key regulator of events associated with prostate cancer metastatic bone disease.

The Role of RUNX2 in Osteosarcoma Oncogenesis.

Osteosarcoma is an aggressive but ill-understood cancer of bone that predominantly affects adolescents. Its rarity and biological heterogeneity have limited studies of its molecular basis. In recent years, an important role has emerged for the RUNX2 “platform protein” in osteosarcoma oncogenesis. RUNX proteins are DNA-binding transcription factors that regulate the expression of multiple genes involved in cellular differentiation and cell-cycle progression. RUNX2 is genetically essential for developing bone and osteoblast maturation. Studies of osteosarcoma tumours have revealed that the RUNX2 DNA copy number together with RNA and protein levels are highly elevated in osteosarcoma tumors. The protein is also important for metastatic bone disease of prostate and breast cancers, while RUNX2 may have both tumor suppressive and oncogenic roles in bone morphogenesis. This paper provides a synopsis of the current understanding of the functions of RUNX2 and its potential role in osteosarcoma and suggests directions for future study.

Bone tissue-specific transcription of the osteocalcin gene: Role of an activator osteoblast-specific complex and suppressor Hox proteins that bind the OC box

Bone‐specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue‐specific expression. By gel mobility shift assays with wild‐type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence‐specific interactions with the primary transcriptional element, the OC box (nt −99 to −76). This OC box binding protein (OCBP) is present only in osteoblast‐like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2–3‐fold increase in transcription, while decreased binding results in transcription 30–40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non‐osseous cell lines. In summary, we demonstrate association of at least two classes of tissue‐restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.

Contributions of Nuclear Architecture to Transcriptional Control

Three parameters of nuclear structure contribute to transcriptional control. The linear representation of promoter elements provides competency for physiological responsiveness within the contexts of development as well as cycle- and phenotype-dependent regulation. Chromatin structure and nucleosome organization reduce distances between independent regulatory elements providing a basis for integrating components of transcriptional control. The nuclear matrix supports gene expression by imposing physical constraints on chromatin related to three-dimensional genomic organization. In addition, the nuclear matrix facilitates gene localization as well as the concentration and targeting of transcription factors. Several lines of evidence are presented that are consistent with involvement of multiple levels of nuclear architecture in cell growth and tissue-specific gene expression during differentiation. Growth factor and steroid hormone responsive modifications in chromatin structure, nucleosome organization, and the nuclear matrix that influence transcription of the cell cycle-regulated histone gene and the bone tissue-specific osteocalcin gene during progressive expression of the osteoblast phenotype are considered.

Phosphorylation of the oncogenic transcription factor interferon regulatory factor 2 (IRF2) in vitro and in vivo

IRF2 is a transcription factor, possessing oncogenic potential, responsible for both the repression of growth‐inhibiting genes (interferon) and the activation of cell cycle‐regulated genes (histone H4). Surprisingly little is known about the post‐translational modification of this factor. In this study, we analyze the phosphorylation of IRF2 both in vivo and in vitro. Immunoprecipitation of HA‐tagged IRF2 expressed in 32P‐phosphate labelled COS‐7 cells demonstrates that IRF2 is phosphorylated in vivo. Amino acid sequence analysis reveals that several potential phosphorylation sites exist for a variety of serine/threonine protein kinases, including those of the mitogen activated protein (MAP) kinase family. Using a battery of these protein kinases we show that recombinant IRF2 is a substrate for protein kinase A (PKA), protein kinase C (PKC), and casein kinase II (CK2) in vitro. However, other serine/threonine protein kinases, including the MAP kinases JNK1, p38, and ERK2, do not phosphorylate IRF2. Two‐dimensional phosphopeptide mapping of the sites phosphorylated by PKA, PKC, and CKII in vitro demonstrates that these enzymes are capable of phosphorylating IRF2 at multiple distinct sites. Phosphoaminoacid analysis of HA‐tagged IRF2 immunoprecipitated from an asynchronous population of proliferating, metabolically phosphate‐labelled cells indicates that this protein is phosphorylated exclusively upon serine residues in vivo. These results suggest that the oncogenic protein IRF2 may be regulated via multiple pathways during cellular growth. J. Cell. Biochem. 66:175‐183, 1997.

Nuclear microenvironments: an architectural platform for the convergence and integration of transcriptional regulatory signals

Functional interrelationships between the intranuclear organization of nucleic acids and regulatory proteins are obligatory for fidelity of transcriptional activation and repression. In this article, using the Runx/AML/Cbfa transcription factors as a paradigm for linkage between nuclear structure and gene expression we present an overview of growing insight into the dynamic organization and assembly of regulatory machinery for gene expression at microenvironments within the nucleus. We address contributions of nuclear microenvironments to the convergence and integration of regulatory signals that mediate transcription by supporting the combinatorial assembly of regulatory complexes.

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: How to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24°C, 38°C and 55°C) were investigated. To avoid this problem in microspores, two incubation temperatures (38°C and 55°C) at different periods after GUS assay (4, 24 and 48h) and in the presence of 1mM potassium ferricyanide and 1mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1mM potassium ferricyanide and 1mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55°C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2μM gibberellic acid.

Interrelationships Between Nuclear Structure and Transcriptional Control of Cell Cycle and Tissue-Specific Genes

Three parameters of nuclear structure contribute to transcriptional control. The linear representation of promoter elements provides competency for physiological responsiveness within the contexts of developmental as well as cell cycle and phenotype-dependent regulation. Chromatin structure and nucleosome organization reduce distances between independent regulatory elements providing a basis for integrating components of transcriptional control. The nuclear matrix supports gene expression by imposing physical constraints on chromatin related to three dimensional genomic organization. In addition, the nuclear matrix facilitates gene localization as well as the concentration and targeting of transcription factors. Several lines of evidence are presented which are consistent with involvement of multiple levels of nuclear architecture in cell growth and tissue-specific gene expression during differentiation. Growth factor and steroid hormone responsive modifications in chromatin structure, nucleosome organization and the nuclear matrix are considered which influence transcription of the cell cycle regulated histone gene and the bone tissue-specific osteocalcin gene during progressive expression of the osteoblast phenotype.

Erratum: Bipartite structure of the proximal promoter of a human H4 histone gene (Journal of Cellular Biochemistry (1995) 58 (372-379))

The proximal promoter of the human H4 histone gene FO108 contains two regions of in vivo protein‐DNA interaction, Sites I and II. electrophoretic, mobility shift assays using a radiolabeled DNA probe revealed that several proteins present in HeLa cell nuclear extracts bound specifically to Site 1 (nt‐125 to nt‐86). The most prominent complex, designated HiNF‐C, and a complex of greater mobility, HiNF‐C′, using were specifically competed by an Sp1 consensus oligonucleotide. Fractionation of HiNF‐C using wheat germ agglutinin affinity chromatography suggested that, like Sp1, HiNF‐C contains N‐acetylglucosamine moieties. Two minor complexes of even greater mobility, designated HiNF‐E and F, were competed by ATF consensus oligonucleotides. A DNA probe carrying a site‐specific mutation in the distal portion of Site I failed to bind HiNF‐E, indicating that this protein associated specifically to this region. UV cross‐linking analysis showed that several proteins of different molecular wieghts interact specifically with Site I. These data indicate that Site I possesses as bipartite structure and that multiple proteins present in HeLa cell nuclear extracts interact specifically with Site I sequences.