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The article presents a study which examined human primary osteocytes embedded throughout the mineralized matrix in a three-dimensional (3D) culture method. The method was used to study the osteoblast-osteocyte differentiation pathway and the osteocyte interaction with the other bone cells. It is stated that histological analysis and immunohistochemistry revealed cell proliferation with an anti-human Ki67 antigen antibody.

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. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Investigates the global role of protein kinase CK2 in chromatin remodeling. Analysis of the involvement of CK2 in gene expression at cell cycle; Levels of cellular organization; Components of stress signaling, growth signaling, and survival signaling pathways.

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. [Copyright &y& Elsevier]

Sites essential for tight physical intersubunit interaction in protein kinase CK2, a tetramer composed of two catalytic (α and/or α′) and two regulatory subunits (β), have been assigned to the C-terminal part of subunit β. Mutational analysis suggests region 171-181 of β to be one of these but this is not consistent with the observation of coprecipitation of catalytic subunits by antibodies directed specifically to this β segment which indicates that this region is accessible to antibodies even if the β subunit is associated with the α subunit. In an attempt to clarify the apparent contradiction, we have subdivided β-(155-181)-peptide, which includes the fragment of β and that both binds to catalytic subunits and stimulates kinase activity, into six more or less overlapping peptides with a length of 9-16 amino acid residues and performed peptide competition and α subunit binding assays. The kinase-stimulating effect of β-(155-181)-peptide was counteracted significantly by β-(162-175)-peptide while the others had less or no effect. In an ELISA assay with the peptides covalently bound to wells via a C10 spacer arm, binding of α occurred besides β-(155-181)-peptide only with β-(162-175)-peptide and β-(165-175)-peptide. The data provide the first evidence that the contact and the stimulation competences of subunit β represent separable functions and suggest further that a tight physical contact to catalytic subunits is located at position 162-175 consisting of a hydrophobic stretch (position 162-171) and a hydrophilic binding motif, PEY (position 172-174), with the adjoining downstream part allowing for surface exposure and antibody binding. The presence of several Pro residues within that region might relate to the structural basis for both the α-β interaction and the surface orientation. [ABSTRACT FROM AUTHOR]

Human casein kinase II (CKII) is a ubiquitous and multipotential Ser/Thr kinase involved in Ihe regulation of cell growth and differentiation. Biochemically, two characteristics are particularly notable; first, the tetrameric composition of two catalytic subunits (α and/or α′) and two regulatory subunits (β); second, the autophosphorylation of the holoenzyme at the N-terminus of CKIIβ, suspected to be involved in tuning of the kinase activity. Whether CKIIα and CKIIα′ reconstitute comparably with CKIIβ to form holoenzyme is unclear. For a systematic investigation, the complete set of recombinant CKII subunits and of autophosphorylation mutants of CKIIβ were expressed in Escherichia coli and comparative reconstitutions carried out. At 1:1 molar ratio, CKIIβ stimulated both catalytic subunits roughly fivefold with phosvitin as a substrate. The level of activity reached with both of the reconstituted CKII isoforms was of the same order of magnitude as that of holoenzyme isolated from human placenta. It was also similar to a recombinant α2β2 holoenzyme whose expression had been attained in E. coli with a bicistronic construct containing the coding regions of CKIIβ and CKIIα in a tandem arrangement. Both Ser2 and Ser3 were identified as the autophosphorylation sites; replacement of one of these with Ala by oligonucleotide-mediated site-directed mutagenesis influenced only the extent of CKIIβ autophospborylation, replacement of both resulted in a loss of autophosphorylation. Despite these differences, the stimulatory effect of all the CKIIβ mutants was comparable both to each other and to that of wild-type CKIIα′. This was also obtained when substrates other than phosvitin were employed such as tubulin, or upstream-binding factor (UBF). However, the degree of stimulation was substrate specific and ranged from 2--5-fold with no major differences between CKIIα and CKIIα′ stimulation. Calmodulin phosphorylation by both CKIIα and CKIIα′ was decreased similarly by CKIIβ and the CKIIβ mutants. Proteins such as cAMP-responsive-element-binding protein (CREB), HPV16 E7 or Jun were not phosphorylated by either catalytic subunit but became substrates of both in the presence of CKIIβ or CKIIβ mutants. The data suggest that CKIIα and CKIIα′ form similar CKII holoenzymes and that the tuning of holoenzyme activity is independent of the autopbosphorylation status of CKIIβ. [ABSTRACT FROM AUTHOR]

Recently we demonstrated the presence of cell-surface-located cAMP-dependent protein kinase (ecto-PK A) activity in a number of different cel types [Kübler, D., Pyerin, W., Bill, O., Hotz, A., Sonka, J. and Kinzel, V. (1989). J. Biol. Chem. 264, 14549-14555]. The question of the physiological role of externally directed kinase activity prompted a search for potential natural substrates present in the intercellular fluid. In the present study we have investigated the phosphorylation by ecto-PK A of the human atrial natriuretic peptide ANP99-126, a hormone released by cardiac cells. This 28-amino-acid peptide carries the phosphorylation consensus sequence Arg-Arg-Ser-Ser for the PK A. Incubation of various cell lines (including epithelial, epidermal, myoblast and lymphoma cells) or freshly isolated blood cells (macrophages, erythrocytes and platelets) with ANP in the presence of low micromolar concentrations of ATP resulted in the phosphorylation of ANP at Ser residues. The ANP phosphorylation reaction proved strictly dependent on cAMP; cAMP could not be replaced by cGMP. The phosphorylation was inhibited by the PK A-specific inhibitory peptide and increased linearily for up to 15 min and with a Km value of 3-5 µM for ANP. At higher ATP concentrations (>100 µM) the incorporation rates amounted to about 0.3 mmol P (mol ANP)-1min1. The rise of intracellular cAMP in HEL30 (an epidermal cell line) after application of the β0adrenergic receptor agonist isoproterenol led to an approximately three-fold stimulation of ANP phosphorylation which appears to be brought about by an efflux of intracellular cAMP. Employing cell supernatant fluids and cell sonicates, it could be shown that the phosphorylation of ANP results from the ecto-PK A. Comparison of ANP with ANP phosphorylated in vitro using purified catalytic subunit of PK A. showed that phosphorylation is accompanied by certain changes in the average solution conformation of the peptide, consistent with the changes known to occur in its biological activity. Our results demonstrate cAMP-dependent phosphorylation of the peptide hormone analogue ANP99-126 by intact cells through ecto-PK A, an intriguing mechanism for post-translational processing of ANP. [ABSTRACT FROM AUTHOR]

Phosvitin/casein kinase type II is an ubiquitous, highly conserved enzyme consisting of subunits α, α′ and β. Subunit β, presumably serving regulatory functions, was prepared from human placenta and the amino acid sequence of a protease digestion peptide was determined. The deduced nucleotide sequence was employed for the synthesis of a mixture of 20mers as a hybridization probe to screen a λgt10 HeLa cell cDNA library for clones encoding subunit β. A full-length clone consisting of 1013 bp was isolated and the sequence of both strands determined. The deduced amino add sequence of the largest open reading frame encodes 215 amino acid residues predicting a maximum Mr of 24925. The nucleotide sequence of the human β subunit shows a similarity of 75% to that of Drosophila melanogaster and the deduced amino acid sequence a similarity of 88% and 97% to that of D. melanogaster and bovine lung, respectively. No further protein sequence currently known exhibits any notable similarity. Using a 537-bp restriction fragment of the cDNA clone representing 80% of the coding region, a 1-kb subunit-β transcript was detected by Northern hybridization in total RNA prepared from human epithelial cells and placenta as well as from bovine heart suggesting a single transcript of the β-subunit gene and also demonstrating high similarity also between the human and bovine nucleotide sequences. The same restriction fragment was used as the probe to indicate in Southern hybridizations that the corresponding genotnic DNA contains at least one intron of roughly 2.5 kb in length and presumably is a single copy gene. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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.

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.

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.

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.