Hilary A. Feister

Nuclear Matrix Proteins and Osteoblast Gene Expression

The molecular mechanisms that couple osteoblast structure and gene expression are emerging from recent studies on the bone extracellular matrix, integrins, the cytoskeleton, and the nucleoskeleton (nuclear matrix). These proteins form a dynamic structural network, the tissue matrix, that physically links the genes with the substructure of the cell and its substrate. The molecular analog of cell structure is the geometry of the promoter. The degree of supercoiling and bending of promoter DNA can regulate transcriptional activity. Nuclear matrix proteins may render a change in cytoskeletal organization into a bend or twist in the promoter of target genes. We review the role of nuclear matrix proteins in the regulation of gene expression with special emphasis on osseous tissue. Nuclear matrix proteins bind to the osteocalcin and type I collagen promoters in osteoblasts. One such protein is Cbfa1, a recently described transcriptional activator of osteoblast differentiation. Although their mechanisms of action are unknown, some nuclear matrix proteins may act as “architectural” transcription factors, regulating gene expression by bending the promoter and altering the interactions between other trans‐acting proteins. The osteoblast nuclear matrix is comprised of cell‐ and phenotype‐specific proteins including proteins common to all cells. Nuclear matrix proteins specific to the osteoblast developmental stage and proteins that distinguish osteosarcoma from the osteoblast have been identified. Recent studies indicating that nuclear matrix proteins mediate bone cell response to parathyroid hormone and vitamin D are discussed.

NP/NMP4 transcription factors have distinct osteoblast nuclear matrix subdomains.

The mechanisms underlying the coupling of type I collagen and matrix metalloproteinase (MMP) expression to cell structure and adhesion are poorly understood. We propose that nuclear matrix architectural transcription factors link cell structure and transcription via their association with nuclear matrix subdomains and by their capacity for altering promoter geometry. NP/NMP4 are nuclear matrix proteins that contain from five to eight Cys2His2 zinc fingers. Some NP/NMP4 isoforms bind to the rat type I collagen α1(I) polypeptide chain promoter in the manner of architectural transcription factors and alter basal transcription in osteoblast‐like cells (Thunyakitpisal et al. in review). Certain isoforms of NP/NMP4 are identical to CIZ, Cas‐interacting zinc finger protein, a nucleocytoplasmic shuttling protein that associates with focal adhesions and regulates MMP expression [Nakamoto et al. (2000): Mol Cell Biol 20:1649–1658]. To better understand the role of subnuclear architecture in collagen and MMP expression, we mapped the osteoblast nuclear distribution of NP/NMP4 proteins and identified the functional motifs necessary for nuclear localization and nuclear matrix targeting. Immunofluorescence microscopy was used to determine the cellular and subnuclear distribution of native NP/NMP4 proteins and green fluorescent protein (GFP)‐NP/NMP4 fusion proteins in osteoblast‐like cells. All GFP‐NP/NMP4 fusion proteins localized to the nucleus, but accumulated in distinct nuclear matrix subdomains. The zinc finger domain was necessary and sufficient for nuclear import and matrix targeting. We conclude that the arrangement of the NP/NMP4 zinc fingers largely determines the subnuclear location of these isoforms. J. Cell. Biochem. 79:506–517, 2000.

The expression of the nuclear matrix proteins NuMA, topoisomerase II-α, and -β in bone and osseous cell culture: Regulation by parathyroid hormone

Bone cells undergo changes in cell structure during phenotypic development. Parathyroid hormone (PTH) induces a change in osteoblast shape, a determinant of collagen expression. We hypothesize that alterations in bone cell shape reflect and direct gene expression as governed, in part, by nuclear organization. In this study, we determined whether the expression of nuclear matrix proteins that mediate nuclear architecture, NuMA, topoisomerase II (topo II)-α, and -β, were altered during osteoblast development and response to PTH in vivo. NuMA forms an interphase nuclear scaffold in some cells, the absence of which may accommodate alterations in nuclear organization necessary for specific functions. Topo II enzymes are expressed in bone cells; the α-isoform is specific to proliferating cells. We used immunohistochemistry and flow cytometry to determine whether NuMA is expressed in the primary spongiosa of the rat metaphyseal femur and whether expression of NuMA, topo II-α, and II-β changes during osteoblast development or with PTH treatment. NuMA and topo II-β were expressed in marrow cells, osteoblasts, osteocytes, and chondrocytes. These proteins were not detected in osteoclasts in vivo, but were observed in cultured cells. Bone marrow cells expressed topo II-α. All three proteins were expressed in cultures of rat osteoblast-like UMR-106 cells. PTH treatment downregulated the number of topo II-α-immunopositive cells, correlated with a decrease in S-phase cells, in both bone tissue and cell culture. We conclude that, in vivo, nuclear matrix composition is altered during bone cell development and that anabolic doses of PTH attenuate the proliferative capacity of osteogenic cells, in part, by targeting topo II-α expression.

Parathyroid hormone regulates the expression of rat osteoblast and osteosarcoma nuclear matrix proteins

Parathyroid hormone (PTH) alters osteoblast morphology. How these changes in cell shape modify nuclear structure and ultimately gene expression is not known. Chronic exposure to rat PTH (1–34) [10 nM] attenuated the expression of 200, 190, and 160 kD proteins in the nuclear matrix‐intermediate filament subfraction of the rat osteosarcoma cells, ROS 17/2.8 [Bidwell et al. (1994b): Endocrinology 134:1738–1744]. Here, we determined that these same PTH‐responsive proteins were expressed in rat metaphyseal osteoblasts. We identified the 200 kD protein as a non‐muscle myosin. Although the molecular weights, subcellular distribution, and half‐lives of the 190 and 160 kD proteins were similar to topoisomerase II‐α and ‐β, nuclear matrix enzymes that mediate DNA topology, the 190 and 160 kD proteins did not interact with topoisomerase antibodies. Nevertheless, the expression of topoisomerase II‐α, and NuMA, a component of the nuclear core filaments, was also regulated by PTH in the osteosarcoma cells. The 190 kD protein was selectively expressed in bone cells as it was not observed in OK opossum kidney cells, H4 hepatoma cells, or NIH3T3 cells. PTH attenuated mRNA expression of the PTH receptor in our cell preparations. These results demonstrate that PTH selectively alters the expression of osteoblast membrane, cytoskeletal, and nucleoskeletal proteins. Topoisomerase II‐α, NuMA, and the 190 and 160 kD proteins may direct the nuclear PTH signalling pathways to the target genes and play a structural role in osteoblast gene expression.

Parathyroid Hormone Regulates the Expression of the Nuclear Mitotic Apparatus Protein in the Osteoblast-like Cells, ROS 17/2.8

The parathyroid hormone (PTH) signaling pathways that effect changes in osteoblast gene expression also alter the organization of the cytoskeletal proteins. PTH regulates the expression of nucleoskeletal proteins, such as nuclear mitotic apparatus protein (NuMA) and topoisomerase II-alpha. NuMA is a structural component of the interphase nucleus and organizes the microtubules of the mitotic spindle during mitogenesis. We propose that PTH-induced alterations in osteoblast cytoarchitecture are accompanied by changes in osteoblast nuclear structure that contribute to changes in gene expression. We used immunofluorescence and confocal microscopy to determine the effect of PTH on the expression and nuclear distribution of NuMA in the rat osteosarcoma cell line, ROS 17/2.8. Cells were treated with PTH or vehicle, then fixed and stained with NuMA antibody. Optical sections of interphase naive cells revealed a diffuse distribution of NuMA, interspersed with speckles, in the central nuclear planes but not in nucleoli. During the metaphase and anaphase, NuMA localized at the mitotic spindle apparatus. The percentage of NuMA-immunopositive ROS 17/2.8 cells decreased with increasing confluence, but serum starvation did not attenuate NuMA expression. Cell density-dependent changes in cytoskeletal organization were observed in these cells. PTH treatment induced changes in cytoskeletal organization and increased the percentage of NuMA-immunopositive ROS 17/2.8 cells. These data suggest that PTH effects changes in osteoblast nuclear architecture by regulating NuMA, and that these alterations may be coupled to cytoskeletal organization.

TISSUE MATRIX PROTEIN EXPRESSION IN HUMAN OSTEOBLASTS, OSTEOSARCOMA TUMORS, AND OSTEOSARCOMA CELL LINES

Treatment for osteosarcoma is problematic because there are no prognostic markers. Diagnosis is primarily limited to cytologic grading. Oncogenesis alters cell structure therefore osteoblast tissue matrix proteins (extracellular matrix, cytoskeletal, intermediate filament, and nuclear matrix proteins), components of the cell substructure, are candidates for osteosarcoma markers. Structural proteins of the extracellular matrix, e.g. the collagens, are useful for diagnosis but not for tumors that produce little osteoid. To identify principal cellular tissue matrix proteins that distinguish normal from transformed human osteoblasts, their expression in normal osteoblasts, two osteosarcoma cell lines, and three primary osteosarcoma tumors were compared. The tumors were graded as (i) intermediate, (ii) high, and (iii) high grade recurrent. The 1-D SDS/PAGE profiles of the major components of the nuclear matrix and intermediate filament fractions from normal osteoblasts did not vary with biopsy site, age, or sex of patients. These profiles included known cytoskeletal proteins and OB250, a ∼250 kD protein(s) observed in the intermediate filament fraction. A loss of protein bands, including OB250, was observed in the osteosarcoma cell lines and tumors. The intermediate and high grade tumors exhibited nearly identical protein profiles including potential tumor-specific proteins and collagen, consistent with the presence of intracellular collagen fibers in osteosarcoma. A microsequence was obtained for OT25, a novel low molecular weight protein observed in osteosarcoma cell lines. Fibrinogen γ-chain, a protein that mediates cell adhesion was recovered from the high grade recurrent tumor.

Topoisomerase II expression in osseous tissue

The molecular mechanisms that mediate the transition from an osteoprogenitor cell to a differentiated osteoblast are unknown. We propose that topoisomerase II (topo II) enzymes, nuclear proteins that mediate DNA topology, contribute to coordinating the loss of osteoprogenitor proliferative capacity with the onset of differentiation. The isoforms topo II‐α and ‐β, are differentially expressed in nonosseous tissues. Topo II‐α expression is cell cycle‐dependent and upregulated during mitogenesis. Topo II‐β is expressed throughout the cell cycle and upregulated when cells have plateaued in growth. To determine whether topo II‐α and ‐β are expressed in normal bone, we analyzed rat lumbar vertebrae using immunohistochemical staining. In the tissue sections, topo II‐α was expressed in the marrow cavity of the primary spongiosa. Mature osteoblasts along the trabecular surfaces did not express topo II‐α, but were immunopositive for topo II‐β, as were cells of the marrow cavity. Confocal laser scanning microscopy was used to determine the nuclear distribution of topo II in rat osteoblasts isolated from the metaphyseal distal femur and the rat osteosarcoma cells, ROS 17/2.8. Topo II‐α exhibited a punctate nuclear distribution in the bone cells. Topo II‐β was dispersed throughout the interior of the nucleus but concentrated at the nuclear envelope. Serum starvation of the cells attenuated topo II‐α expression but did not modulate expression of the β‐isoform. These results indicate that the loss of osteogenic proliferation correlates with the downregulation of topo II‐α expression. J. Cell. Biochem. 67:451–465, 1997.