Human primary osteocyte differentiation in a 3D culture system.

Studies on primary osteocytes, which compose >90-95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 3D culture method of primary human osteoblast differentiation into osteocytes. These 3D-differentiated osteocytes were compared with 2D-cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times, as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Histological analysis showed, after only 1 wk, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte-like cells. Real-time PCR expression of a set of bone-related genes confirmed their osteocyte phenotype. Comparison with plastic-cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast-osteocyte differentiation pathway, the osteocyte interaction with the other bone cells, and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the cross-talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.

Lack of noggin expression by cancer cells is a determinant of the osteoblast response in bone metastases.

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

The catalytic subunit alpha' gene of human protein kinase CK2 (CSNK2A2): genomic organization, promoter identification and determination of Ets1 as a key regulator.

The human genome contains four protein kinase CK2 loci, enclosing three active genes coding for the catalytic subunits alpha and alpha' and the regulatory subunit beta, and a processed alpha subunit pseudogene. Extensive structure and transcriptional control data of the genes are available, except for the CK2alpha' gene (CSNK2A2). Using in silico and experimental approaches, we find CSNK2A2 to be located on the long arm of chromosome 16 (in contrast to published data), to span 40kb and to consist of 12 exons, with the translational start in Exon 1 and the stop in Exon 11. Exon/intron boundaries conform to the gt/ag rule, and various potential polyadenylation signals determine transcript species with lengths of 1.7-5.7 kb. The upstream region of the gene displays housekeeping characteristics, lacking a TATA box and possessing several GC boxes as well as a CpG island around Exon 1. According to reporter gene assay results, the promoter activity ranges from -1308 to 197 with the highest activity in region -396 to -129. This region contains binding motifs for various transcription factors, including NFkappaB, Sp and Ets family members. Site-directed mutagenesis indicates that the Ets motifs play, in cooperation with Sp motif clusters, a central role in regulating CK2alpha' gene transcription. A similar control has been described for the transcription of the CK2alpha and CK2beta genes so that the presented data are compatible with the assumption of a coordinate transcriptional regulation of all three active human CK2 genes decisively determined by Ets family members.

Protein kinase CK2 in gene control at cell cycle entry.

Protein kinase CK2 has diverse links to gene control and cell cycle. Comparative genome-wide expression profiling of CK2 mutants of the budding yeast Saccharomyces cerevisiae at cell cycle entry has revealed that a significant proportion of cell-cycle genes are affected by CK2. Here, we examine how CK2 realizes this effect. We show that the CK2 action may be directed to gene promoters causing genes with promoter homologies to respond comparably to CK2 perturbation. Examples are metabolic pathway and nutrition supply genes such as the PHO and MET regulon genes, responsible for phosphate maintenance and methionine biosynthesis, respectively. CK2 perturbation affects both regulons permanently and both via repression of a central transcription factor, but with different mechanisms: In the PHO regulon, the gene encoding the central transcription factor Pho4 is repressed and, in addition, Pho4 and/or the cyclin-dependent kinase of the regulon's control complex may be affected by CK2 phosphorylation. In the MET regulon, the repression of the central transcription factor Met4 occurs not by expression inhibition, but rather by availability tuning via a CK2-mediated phosphorylation of a degradation complex. On the other hand, the CK2 action may be directed to the chromatin regulon, thus affecting globally the expression of genes, i.e., the CK2 perturbation results either in comparable responses of genes which have no promoter homologies or in deviating responses despite promoter homologies. The effect is rather transient and concerns aside various cell cycle control genes a notable number of genes encoding chromatin remodeling and modification proteins with functions in chromatin assembly and (anti-)silencing as well as in histone (de-)acetylation, and frequently are also substrates of CK2, suggesting additional tuning at protein level. In line with these findings, we observe in human cells sequence-independent but cell-cycle-dependent CK2 associations with promoters of cell-cycle-regulated genes at periods of extensive gene expression alterations, including cell cycle entry. Our observations are compatible with the idea that the gene control by CK2 is achieved via different mechanisms and at different levels of organization and includes a global role in transcription-related chromatin remodelling and modification.

Reduction of pain and fracture incidence after kyphoplasty: 1-year outcomes of a prospective controlled trial of patients with primary osteoporosis.

Previously, we reported significantly reduced pain and improved mobility persisting for 6 months after kyphoplasty of chronically painful osteoporotic vertebral fractures in the first prospective controlled trial. Since improvement of spinal biomechanics by restoration of vertebral morphology may affect the incidence of fracture, long-term clinical benefit and thereby cost-effectiveness, here we extend our previous work to assess occurrence of new vertebral fractures and clinical parameters 1 year after kyphoplasty compared with a conservatively treated control group. Sixty patients with osteoporotic vertebral fractures due to primary osteoporosis were included: 40 patients were treated with kyphoplasty, 20 served as controls. All patients received standard medical treatment. Morphological characteristics, new vertebral fractures, pain (visual analog scale), physical function [European Vertebral Osteoporosis Study (EVOS) score] (range 0-100 each) and back-pain-related doctors' visits were re-assessed 12 months after kyphoplasty. There were significantly fewer patients with new vertebral fractures of the thoracic and lumbar spine, after 12-months, in the kyphoplasty group than in the control group (P=0.0084). Pain scores improved from 26.2 to 44.4 in the kyphoplasty group and changed from 33.6 to 34.3 in the control group (P=0.008). Kyphoplasty treated patients required a mean of 5.3 back-pain-related doctors' visits per patient compared with 11.6 in the control group during 12 months follow-up (P=0.006). Kyphoplasty as an addition to medical treatment and when performed in appropriately selected patients by an interdisciplinary team persistently improves pain and reduces occurrence of new vertebral fractures and healthcare utilization for at least 12 months in individuals with primary osteoporosis.

Treatment of painful vertebral fractures by kyphoplasty in patients with primary osteoporosis: a prospective nonrandomized controlled study.

UNLABELLED: This study investigates the effects of kyphoplasty on pain and mobility in patients with osteoporosis and painful vertebral fractures compared with conventional medical management. INTRODUCTION: Pharmacological treatment of patients with primary osteoporosis does not prevent pain and impaired activity of patients with painful vertebral fractures. Therefore, we evaluated the clinical outcome after kyphoplasty in patients with vertebral fractures and associated chronic pain for >12 months. MATERIALS AND METHODS: Sixty patients with primary osteoporosis and painful vertebral fractures presenting for >12 months were included in this prospective, nonrandomized controlled study. Twenty-four hours before performing kyphoplasty, the patients self-determined their inclusion into the kyphoplasty or control group so that 40 patients were treated with kyphoplasty, whereas 20 served as controls. This study assessed changes in radiomorphology, pain visual analog scale (VAS) score, daily activities (European Vertebral Osteoporosis Study [EVOS] score), number of new vertebral fractures, and health care use. Outcomes were assessed before treatment and at 3 and 6 months of follow-up. All patients received standard medical treatment (1g calcium, 1000 IE vitamin D(3), standard dose of oral aminobisphosphonate, pain medication, physical therapy). RESULTS: Kyphoplasty increased midline vertebral height of the treated vertebral bodies by 12.1%, whereas in the control group, vertebral height decreased by 8.2% (p = 0.001). Augmentation and internal stabilization by kyphoplasty resulted in a reduction of back pain. VAS pain scores improved in the kyphoplasty group from 26.2 +/- 2 to 44.2 +/- 3.3 (SD; p = 0.007) and in the control group from 33.6 +/- 4.1 to 35.6 +/- 4.1 (not significant), whereas the EVOS score increased in the kyphoplasty group from 43.8 +/- 2.4 to 54.5 +/- 2.7 (p = 0.031) and in the control group from 39.8 +/- 4.5 to 43.8 +/- 4.6 (not significant). The number of back pain-related doctor visits within the 6-month follow-up period decreased significantly after kyphoplasty compared with controls: mean of 3.3 visits/patient in the kyphoplasty group and a mean of 8.6 visits/patient in the control group (p = 0.0147). CONCLUSIONS: The results of this study show significantly increased vertebral height, reduced pain, and improved mobility in patients after kyphoplasty. Kyphoplasty performed in appropriately selected osteoporotic patients with painful vertebral fractures is a promising addition to current medical treatment.

Cystic duct dilatations and proliferative epithelial lesions in mouse mammary glands upon keratin 5 promoter-driven overexpression of cyclooxygenase-2.

Expression and pharmacological studies support a contribution of cyclooxygenase (COX)-2 to mammary gland tumorigenesis. In a recent transgenic study, mouse mammary tumor virus promoter-driven COX-2 expression in mouse mammary glands was shown to result in alveolar hyperplasia, dysplasia, and carcinomas after multiple rounds of pregnancy and lactation. In the study presented here, the effects of constitutive COX-2 overexpression in keratin 5-positive myoepithelial and luminal cells, driven by the keratin 5 promoter in a hormone-independent manner, was investigated. In nulliparous female mice, aberrant COX-2 overexpression correlated with increased prostaglandin (PG) E(2) levels and caused cystic duct dilatations, adenosis, and fibrosis whereas carcinomas developed rarely. This phenotype depended on COX-2-mediated PGE(2) synthesis and correlated with increased expression of proliferation-associated Ki67 in epithelial cells. No changes in the expression of apoptosis-related Bcl-2, caspase 3, or p53 were observed. Hyperproliferation of the mammary gland epithelial cells was associated with increased aromatase mRNA levels in this tissue. The spontaneous pathologies bear analogies to the human breast with fibrocystic changes. Intriguingly, strong COX-2 expression was observed in fibrocystic changes, as compared to low expression in normal breast epithelium. These results show for the first time that aberrant COX-2 expression contributes to the development of fibrocystic changes (FC), indicating that COX-2 and COX-2-mediated PG synthesis represent potential targets for the therapy of this most frequent benign disorder of the human breast.

High-quality RNA preparation for transcript profiling of osteocytes from native human bone microdissections.

Osteocytes, the most abundant bone cell type with important roles in tissue maintenance and pathological aberrations such as observed in bone metastases, are enclosed within a highly compact, calcified extracellular matrix. This location complicates analysis in native bone, with the consequence that despite their importance their in vivo molecular physiology is only poorly understood. We have examined the possibility of isolating osteocyte RNA for transcript profiling from native, frozen bone instead of employing the formalin-fixed, paraffin-embedded, decalcified version routinely used in histology, providing chemically modified and highly disintegrated RNAs. Bone tissue was tape-assisted cryosectioned and fixed to glass slides by support of UV-flash-triggered adhesive polymerization followed by quick hematoxylin-eosin staining to generate a guidance image for microdissection. Using an UVa-nitrogen laser, matrix-enclosed osteocytes were either excised and catapulted into RNA preparation vials or freed of accompanying nonosteocyte cellular material. The influences of bone sectioning, staining, and osteocyte capturing procedures on the prepared osteocyte RNAs were analyzed and the method was optimized accordingly. The obtained osteocyte RNAs showed the expected expression pattern of marker genes (reverse transcriptase-polymerase chain reaction), and, following conversion into fluorescent-labeled cDNAs, led to transcript profiles (cDNAchips; 2600 genes) with scatter-graph geometries indicating suitability for high-confidence evaluation. With the approach described here we introduce a methodological way for the characterization of the in vivo molecular physiology of osteocytes by functional genomics.

Bone metastasis: Osteoblasts affect growth and adhesion regulons in prostate tumor cells and provoke osteomimicry.

Bone metastasis is the primary cause of death in human prostate cancer. Disseminated from primary tumor and distributed via the bloodstream, a proportion of prostate carcinoma cells eventually reach the skeleton and develop into metastases, requiring adhesion to inner bone surfaces lined by osteoblasts. The crosstalk of tumor cells with osteoblasts is a critical but poorly characterized step in the metastatic process. Using an in vitro metastasis model system, we have been examining effects of osteoblast-released factors on gene expression of prostate carcinoma cells. Here, we show by large-scale transcript profiling and quantitative RT-PCR that osteoblast-released factors target in particular the proliferation and adhesion regulons of tumor cells. Genes encoding components of the cell-cycle control machinery and connected pathways are predominantly repressed and cell proliferation is slowed down, resembling in vivo observations assumed to render commonly used chemotherapeutic measures ineffective. Genes encoding anchoring junction components are predominantly elevated, and the adhesion properties of tumor cells are altered. Moreover, prostate carcinoma cells are provoked to undergo osteomimicry, i.e., to express bone cell-related genes. The data indicate that the crosstalk with osteoblasts induces expressional changes in prostate carcinoma cells favoring the bone colonization process.

Genome-wide expression screens indicate a global role for protein kinase CK2 in chromatin remodeling.

Protein kinase CK2, a vital, pleiotropic and highly conserved serine/threonine phosphotransferase is involved in transcription-directed signaling, gene control and cell cycle regulation and is suspected to play a role in global processes. Searching for these global roles, we analyzed the involvement of CK2 in gene expression at cell cycle entry by using genome-wide screens. Comparing expression profiles of Saccharomyces cerevisiae wild-type strains with strains with regulatory or catalytic subunits of CK2 deleted, we found significant alterations in the expression of genes at all cell cycle phases and often in a subunit- and isoform-specific manner. Roughly a quarter of the genes known to be regulated by the cell cycle are affected. Functionally, the genes are involved with cell cycle entry, progression and exit, including spindle pole body formation and dynamics. Strikingly, most CK2-affected genes exhibit no common transcriptional control features, and a considerable proportion of temporarily altered genes encodes proteins involved in chromatin remodeling and modification, including chromatin assembly, (anti-)silencing and histone (de-)acetylation. In addition, various metabolic pathway and nutritional supply genes are affected. Our data are compatible with the idea that CK2 acts at different levels of cellular organization and that CK2 has a global role in transcription-related chromatin remodeling.

Perturbation of protein kinase CK2 uncouples executive part of phosphate maintenance pathway from cyclin-CDK control.

The budding yeast Saccharomyces cerevisiae encounters phosphate starvation by the transcription-regulated PHO pathway. We find that genetic perturbation of protein kinase CK2, a conserved tetrameric Ser/Thr phosphotransferase with links to cell cycle and transcription, affects expression of PHO pathway genes in a subunit- and isoform-specific manner. Remarkably, the genes encoding phosphate supplying phosphatases and transporters are significantly repressed, while the genes encoding components of the central pathway regulator complex, a cyclin-dependent kinase (CDK), a cyclin, and a CDK inhibitor, remain unaltered. Thus, perturbation of CK2 uncouples the executive part of the PHO pathway from its cyclin-CDK control complex.

A positive control for the green fluorescent protein-based one-hybrid system.

The yeast one-hybrid system (1H-system) is applied to studies of transcription-linked DNA-protein interactions under in vivo conditions detected by reporter gene expression. By use of a variant of green fluorescent protein (GFP) as a reporter, the new 1H-system generation represents a time- and work-saving alternative to the established HIS3/lacZ-based form. However, hitherto, a positive control proving the functionality of the system has been missing. We designed a corresponding control vector combination by subcloning mouse p53 cDNA and its binding sequence and, to get the system working, modified the distance between cloning site and reporter gene promoter in the reporter vector by site-directed mutagenesis. This provides, for the first time, a positive control vector combination for the GFP 1H-system, crucial for its employment in any DNA-protein interaction studies.