Release of ATP from human osteoblastic cell lines and primary osteoblasts occurs via exocytosis

Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.

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Receptor expression of neuropeptide in human osteoblastic cell lines (SaOS-2, HOS, and MG-63)

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)45653-9 ◽

1997 ◽

Vol 73 ◽

pp. 288

Author(s):

Akifumi Togari ◽

Michitsugu Arai

Keyword(s):

Cell Lines ◽

Receptor Expression ◽

Osteoblastic Cell ◽

Human Osteoblastic Cell

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Estrogen receptor isoform-specific regulation of endogenous gene expression in human osteoblastic cell lines expressing either ER? or ER?

Journal of Cellular Biochemistry ◽

10.1002/jcb.10633 ◽

2003 ◽

Vol 90 (2) ◽

pp. 315-326 ◽

Cited By ~ 105

Author(s):

David G. Monroe ◽

Barbara J. Getz ◽

Steven A. Johnsen ◽

B. Lawrence Riggs ◽

Sundeep Khosla ◽

...

Keyword(s):

Gene Expression ◽

Estrogen Receptor ◽

Cell Lines ◽

Osteoblastic Cell ◽

Endogenous Gene ◽

Human Osteoblastic Cell ◽

Specific Regulation

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Proinflammatory Response of Human Osteoblastic Cell Lines and Osteoblast-Monocyte Interaction upon Infection with Brucella spp.

Infection and Immunity ◽

10.1128/iai.01259-08 ◽

2008 ◽

Vol 77 (3) ◽

pp. 984-995 ◽

Cited By ~ 45

Author(s):

M. Victoria Delpino ◽

Carlos A. Fossati ◽

Pablo C. Baldi

Keyword(s):

Cell Lines ◽

Joint Destruction ◽

Neutralizing Antibody ◽

Osteoblastic Cell ◽

Proinflammatory Response ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Human Osteoblastic Cell ◽

Culture Supernatants

ABSTRACT The ability of Brucella spp. to infect human osteoblasts and the cytokine response of these cells to infection were investigated in vitro. Brucella abortus, B. suis, B. melitensis, and B. canis were able to infect the SaOS-2 and MG-63 osteoblastic cell lines, and the first three species exhibited intracellular replication. B. abortus internalization was not significantly affected by pretreatment of cells with cytochalasin D but was inhibited up to 92% by colchicine. A virB10 mutant of B. abortus could infect but not replicate within osteoblasts, suggesting a role for the type IV secretion system in intracellular survival. Infected osteoblasts produced low levels of chemokines (interleukin-8 [IL-8] and macrophage chemoattractant protein 1 [MCP-1]) and did not produce proinflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor alpha [TNF-α]). However, osteoblasts stimulated with culture supernatants from Brucella-infected human monocytes (THP-1 cell line) produced chemokines at levels 12-fold (MCP-1) to 17-fold (IL-8) higher than those of infected osteoblasts and also produced IL-6. In the inverse experiment, culture supernatants from Brucella-infected osteoblasts induced the production of IL-8, IL-1β, IL-6, and TNF-α by THP-1 cells. The induction of TNF-α and IL-1β was largely due to granulocyte-macrophage colony-stimulating factor produced by infected osteoblasts, as demonstrated by inhibition with a specific neutralizing antibody. This study shows that Brucella can invade and replicate within human osteoblastic cell lines, which can directly and indirectly mount a proinflammatory response. Both phenomena may have a role in the chronic inflammation and bone and joint destruction observed in osteoarticular brucellosis.

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Tadalafil modulates aromatase activity and androgen receptor expression in a human osteoblastic cell in vitro model

Journal of Endocrinological Investigation ◽

10.1007/s40618-015-0344-1 ◽

2015 ◽

Vol 39 (2) ◽

pp. 199-205 ◽

Cited By ~ 7

Author(s):

A. Aversa ◽

S. Fittipaldi ◽

V. M. Bimonte ◽

F. Wannenes ◽

V. Papa ◽

...

Keyword(s):

Androgen Receptor ◽

In Vitro Model ◽

Receptor Expression ◽

Androgen Receptor Expression ◽

Osteoblastic Cell ◽

Aromatase Activity ◽

Human Osteoblastic Cell ◽

Vitro Model

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E11/gp38 Selective Expression in Osteocytes: Regulation by Mechanical Strain and Role in Dendrite Elongation

Molecular and Cellular Biology ◽

10.1128/mcb.02120-05 ◽

2006 ◽

Vol 26 (12) ◽

pp. 4539-4552 ◽

Cited By ~ 170

Author(s):

Keqin Zhang ◽

Cielo Barragan-Adjemian ◽

Ling Ye ◽

Shiva Kotha ◽

Mark Dallas ◽

...

Keyword(s):

Shear Stress ◽

Cell Lines ◽

Mechanical Load ◽

Mechanical Strain ◽

Three Dimensional ◽

Cell Communication ◽

Bone Surface ◽

Cellular Processes ◽

Primary Osteoblasts

ABSTRACT Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.

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Immunomodulatory Potential of Differently-Terminated Ultra-Small Silicon Carbide Nanoparticles

Nanomaterials ◽

10.3390/nano10030573 ◽

2020 ◽

Vol 10 (3) ◽

pp. 573

Author(s):

Tereza Bělinová ◽

Iva Machová ◽

David Beke ◽

Anna Fučíková ◽

Adam Gali ◽

...

Keyword(s):

Silicon Carbide ◽

Cell Line ◽

Metabolic Activity ◽

Immune Cell ◽

Cellular Membrane ◽

Osteoblastic Cell ◽

Osteoblastic Cell Line ◽

Human Osteoblastic Cell ◽

Different Response ◽

Small Nanoparticles

Ultra-small nanoparticles with sizes comparable to those of pores in the cellular membrane possess significant potential for application in the field of biomedicine. Silicon carbide ultra-small nanoparticles with varying surface termination were tested for the biological system represented by different human cells (using a human osteoblastic cell line as the reference system and a monocyte/macrophage cell line as immune cells). The three tested nanoparticle surface terminations resulted in the observation of different effects on cell metabolic activity. These effects were mostly noticeable in cases of monocytic cells, where each type of particle caused a completely different response (‘as-prepared’ particles, i.e., were highly cytotoxic, –OH terminated particles slightly increased the metabolic activity, while –NH2 terminated particles caused an almost doubled metabolic activity) after 24 h of incubation. Subsequently, the release of cytokines from such treated monocytes and their differentiation into activated cells was determined. The results revealed the potential modulation of immune cell behavior following stimulation with particular ultra-small nanoparticles, thus opening up new fields for novel silicon carbide nanoparticle biomedical applications.

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