Janet M. Hock

Six‐Month Daily Administration of Parathyroid Hormone and Parathyroid Hormone—Related Protein Peptides to Adult Ovariectomized Rats Markedly Enhances Bone Mass and Biomechanical Properties: A Comparison of Human Parathyroid Hormone 1–34, Parathyroid Hormone‐Related Protein 1–36, and SDZ‐Parathyroid Hormone 893

Daily administration of parathyroid hormone (PTH) and PTH‐related protein (PTHrP) peptides has been shown to increase bone mass and strength in animals and, for PTH, to increase bone mass in humans. Long‐term direct comparison of multiple members of the PTH/PTHrP family in vivo has not been reported. We therefore selected three PTH/PTHrP molecules for direct comparison in vivo in an adult rat model of postmenopausal osteoporosis: PTH(1‐34), PTHrP(1‐36), and the PTH analog, SDZ‐PTH 893 {Leu8, Asp10, Lys11, Ala16, Gln18, Thr33, Ala34 human PTH 1–34 [hPTH(1‐34)]}. A 6‐month study was performed in which adult (6‐month‐old) vehicle‐treated ovariectomized (OVX) and sham OVX rats were compared with OVX rats receiving 40 μg/kg per day of either PTH(1‐34), PTHrP(1‐36), or PTH‐SDZ‐893. Bone mass, as assessed by ash weight and densitometry, bone histomorphometry, biomechanical properties at trabecular and cortical sites, and indices of bone formation markedly increased in all three PTH/PTHrP peptide‐treated groups as compared with controls. In general, this improvement followed a rank order of SDZ‐PTH‐893 > PTH > PTHrP. The adverse effect profile also was greatest with SDZ‐PTH‐893; these rats developed moderate hypercalcemia, marked renal calcium accumulation, and displayed a 13% mortality. These studies show that PTH(1‐34), PTHrP(1‐36), and PTH‐SDZ‐893 significantly and progressively increase bone mass and bone strength in this rat model of postmenopausal osteoporosis. The adverse effect profile correlates in general terms with efficacy. All three peptides show promise as skeletal anabolic agents. Further studies in humans will be required to define optimal efficacy‐to‐adverse effect ratios and relative efficacy for each peptide in human osteoporosis.

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.

Bone mineral and collagen quality in humeri of ovariectomized cynomolgus monkeys given rhPTH(1-34) for 18 months

A recent study of ovariectomized monkeys, treated with recombinant human parathyroid hormone (rhPTH)(1–34) at 1 or 5 mg/kg/day for 18 months or for 12 months followed by 6 months withdrawal from treatment, showed significant differences in the geometry and histomorphometry of cortical bone of the midshaft humerus. To determine the extent to which the rapid bone turnover and cortical porosity induced by rhPTH(1–34) in ovariectomized monkeys modified mineral content, mineral crystal maturity and collagen maturity (cross‐link distribution) in the cortical periosteal and endosteal regions, cross‐sections of the cortical bone of the mid‐humerus, were examined using Fourier transform infrared imaging (FTIRI). FTIRI analyses demonstrated that rhPTH(1–34) altered bone mineral and collagen properties in a dose‐dependent manner. Mineral crystal maturity and collagen cross‐link ratio (pyridinoline/dehydro‐dihydroxylysinonorleucine) on both endosteal and periosteal surfaces decreased relative to ovariectomized animals, consistent with new bone formation. These changes were partially sustained after withdrawal of the higher dose of rhPTH(1–34), suggesting a prolonged after‐effect on bone properties for at least two bone remodeling cycles. In conclusion, treatment of ovariectomized monkeys with rhPTH(1–34) had significant effects on cortical bone mineral‐to‐matrix ratio, mineral crystal maturity, and collagen cross‐link ratio. These were fully reversible when the 1‐μg rhPTH(1–34) treatment was withdrawn, but only partially reversed when the 5‐μg rhPTH(1–34) dose was withdrawn.

Activation of the p38 MAPK/Akt/ERK1/2 signal pathways is required for the protein stabilization of CDC6 and cyclin D1 in low‐dose arsenite‐induced cell proliferation

Arsenic trioxide (ATO) is a first‐line anti‐cancer agent for acute promyelocytic leukemia, and induces apoptosis in other solid cancer cell lines including breast cancer cells. However, as with arsenites found in drinking water and used as raw materials for wood preservatives, insecticides, and herbicides, low doses of ATO can induce carcinogenesis after long‐term exposure. At 24u2009h after exposure, ATO (0.01–1u2009µM) significantly increased cell proliferation and promoted cell cycle progression from the G1 to S/G2 phases in the non‐tumorigenic MCF10A breast epithelial cell line. The expression of 14 out of 96 cell‐cycle‐associated genes significantly increased, and seven of these genes including cell division cycle 6 (CDC6) and cyclin D1 (CCND1) were closely related to cell cycle progression from G1 to S phase. Low‐dose ATO steadily increased gene transcript and protein levels of both CDC6 and cyclin D1 in a dose‐ and time‐dependent manner. Low‐dose ATO produced reactive oxygen species (ROS), and activated the p38 MAPK, Akt, and ERK1/2 pathways at different time points within 60u2009min. Small molecular inhibitors and siRNAs inhibiting the activation of p38 MAPK, Akt, and ERK1/2 decreased the ATO‐increased expression of CDC6 protein. Inhibiting the activation of Akt and ERK1/2, but not p38 MAPK, decreased the ATO‐induced expression of cyclin D1 protein. This study reports for the first time that p38 MAPK/Akt/ERK1/2 activation is required for the protein stabilization of CDC6 in addition to cyclin D1 in ATO‐induced cell proliferation and cell cycle modulation from G1 to S phase. J. Cell. Biochem. 111: 1546–1555, 2010.

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.

Novel Interactions between FOXM1 and CDC25A Regulate the Cell Cycle

FOXM1 is a critical regulator of the G1/S and G2/M cell cycle transitions, as well as of the mitotic spindle assembly. Previous studies have suggested that FOXM1 regulates CDC25A gene transcription, but the mechanism remains unknown. Here, we provide evidence that FOXM1 directly regulates CDC25A gene transcription via direct promoter binding and indirect activation of E2F-dependent pathways. Prior literature reported that CDC25B and CDC25C activate CDK1/cyclinB complexes in order to enable phosphorylation of FOXM1. It was unknown if CDC25A functions in a similar manner. We report that FOXM1 transcriptional activity is synergistically enhanced when co-expressed with CDC25A. The increase is dependent upon CDK1 phosphorylation of FOXM1 at T600, T611 and T620 residues. We also report a novel protein interaction between FOXM1 and CDC25A via the C-terminus of FOXM1. We demonstrate that the phosphorylation of Thr 600 and Thr 611 residues of FOXM1 enhanced this interaction, and that the interaction is dependent upon CDC25A phosphatase activity. Our work provides novel insight into the underlying mechanisms by which FOXM1 controls the cell cycle through its association with CDC25A.

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.