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Biomedical subjects

Fredrick M Pavalko

Publications and source records attributed to Fredrick M Pavalko.

8 recordsLinked to original sources
PubMed2007

Osteoblasts and osteocytes respond differently to oscillatory and unidirectional fluid flow profiles.

Bone cells subjected to mechanical loading by fluid shear stress undergo significant architectural and biochemical changes. The models of shear stress used to analyze the effects of loading bone cells in vitro include both oscillatory and unidirectional fluid shear profiles. Although the fluid flow profile experienced by cells within bone is most likely oscillatory in nature, to date there have been few direct comparisons of how bone cells respond to these two fluid flow profiles. In this study we evaluated morphologic and biochemical responses to a time course of unidirectional and oscillatory fluid flow in two commonly used bone cell lines, MC3T3-E1 osteoblasts and MLO-Y4 osteocytes. We determined that stress fibers formed and aligned within osteoblasts after 1 h of unidirectional fluid flow, but this response was not observed until greater than 5 h of oscillatory fluid flow. Despite the delay in stress fiber formation, oscillatory and unidirectional fluid flow profiles elicited similar temporal effects on the induction of both cyclooxygenase-2 (Cox-2) and osteopontin protein expression in osteoblasts. Interestingly, MLO-Y4 osteocytes formed organized stress fibers after exposure to 24 h of unidirectional shear stress, while the number of dendritic processes per cell increased along with Cox-2 protein levels after 24 h of oscillatory shear stress. Despite these differences, both flow profiles significantly altered osteopontin levels in MLO-Y4 osteocytes. Together these results demonstrate that the profile of fluid shear can induce significantly different responses from osteoblasts and osteocytes.

3T3 Cells↗
PubMed2006

Disruption of alpha-actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis.

Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of alpha-actinin (ROD-GFP) that competitively displaces endogenous alpha-actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas alpha-actinin-GFP expression protected, cells from TNF-alpha-induced apoptosis. Further investigation revealed that activation of TNF-alpha-induced survival signals, specifically Akt phosphorylation and NF-kappaB activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-alpha-induced apoptosis. Thus we conclude that alpha-actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-alpha-induced survival signaling.

Actinin↗
PubMed2006

HMGB1 expression and release by bone cells.

Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.

Alkaline Phosphatase↗
PubMed2004

Formation of focal adhesions on fibronectin promotes fluid shear stress induction of COX-2 and PGE2 release in MC3T3-E1 osteoblasts.

Mechanical loading of bone is important for the structural integrity of the skeleton and the maintenance of bone mass. Mechanically loading bone generates fluid shear stress (FSS) across the surface of bone cells resulting in the induction of cyclooxygenase-2 (COX-2) and release of prostaglandins, both of which are necessary for mechanically induced bone formation. However, the mechanisms by which cells transduce FSS-induced signals across the membrane and into the cell remain poorly understood. Focal adhesions, which are specialized sites of attachment between cells and the extracellular matrix, play a role in signal transduction and have been proposed to function as mechanosensors. To directly test whether focal adhesions mediate mechanotransduction in bone cells, we inhibited the formation of focal adhesions by 1). culturing MC3T3-E1 osteoblasts on bovine serum albumin (BSA), which does not contain integrin binding sites or by 2). treating cells cultured on fibronectin with soluble Arg-Gly-Asp-Ser (RGDS) peptide to specifically block integrin-fibronectin interactions. We then subjected the cells to FSS and measured COX-2 induction and PGE(2) release. Both COX-2 induction and PGE(2) release in response to FSS were significantly decreased when osteoblasts were treated with soluble RGDS peptide compared with controls. However, RGDS peptide treatment did not affect FSS-induced ERK phosphorylation. Interestingly, osteoblasts cultured on BSA to suppress focal adhesion formation secreted fibronectin and increased focal adhesion formation over time, which correlated with the induction of COX-2 in response to FSS. Together, these results suggest that fibronectin-induced formation of focal adhesions promotes FSS-induced PGE(2) release and upregulation of COX-2 protein.

3T3 Cells↗
PubMed2003

Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules.

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.

Actin Cytoskeleton↗
PubMed2003

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3.

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.

Animals↗
PubMed2003

Fluid shear-induced NFkappaB translocation in osteoblasts is mediated by intracellular calcium release.

Bone formation in response to exogenous mechanical loading is dependent on prostaglandin synthesis by the inducible isoform of cyclooxygenase, COX-2. While several transcription factors target the COX-2 gene, we examined the role of nuclear factor kappa B (NFkappaB) on COX-2 upregulation in osteoblasts in response to fluid shear due to its involvement in immune and inflammatory responses in other cell types. Application of 12 dyn/cm2 laminar flow to MC3T3-E1 osteoblast-like cells resulted in translocation of NFkappaB to the nucleus within 1 h of the onset of shear, with NFkappaB returning to the cytoplasm after 2 h of continuous flow. NFkappaB translocation in response to shear was inhibited by the protease inhibitor, Nalpha-p-tosyl-L-lysine chloromethylketone hydrochloride (TLCK), or a cell-permeant peptide that blocks the nuclear localization sequence (NLS) on NFkappaB. Block of NFkappaB translocation with these inhibitors blocked the shear-induced upregulation of COX-2. We found that disruption of the actin cytoskeleton with cytochalasin D or microtubules with nocodozol did not alter NFkappaB translocation in response to shear. However, addition of the intracellular Ca2+ chelator BAPTA completely blocked NFkappaB translocation. While block of Ca2+ entry with channel blockers failed to inhibit NFkappaB translocation, inhibition of phospholipase C (PLC)-induced intracellular Ca2+ release with the PLC inhibitor U73122 completely abrogated the NFkappaB response to shear. These data indicate that NFkappaB translocation to the nucleus is essential for the fluid shear-induced increase in COX-2. Further, these studies suggest that intracellular Ca2+ release, but not the cytoskeletal architecture, is important to NFkappaB translocation.

Animals↗