Monday, July 11, 2011

Fluid flow: LSJL v osteocytes & osteoblasts v chondrocytes

Since LSJL samples likely included osteocytes in the gene expression analysis. It should be useful to know which genetic upregulation is osteocyte specific.

Pulsating fluid flow modulates gene expression of proteins involved in Wnt signaling pathways in osteocytes.

"MC3T3-E1 osteoblasts were studied as a positive control for the MLO-Y4 osteocyte response to mechanical loading. MLO-Y4 osteocytes and MC3T3-E1 osteoblasts were submitted to 1-h PFF (0.7 +/- 0.3 Pa, 5 Hz), and postincubated (PI) without PFF for 0.5-3 h. Gene expression of proteins related to the Wnt canonical and noncanonical pathways were studied using real-time polymerase chain reaction (PCR). In MLO-Y4 osteocytes, PFF upregulated gene expression of Wnt3a, c-jun, connexin 43, and CD44 at 1-3-h PI. In MC3T3-E1 osteoblasts, PFF downregulated gene expression of Wnt5a and c-jun at 0.5-3-h PI. In MLO-Y4 osteocytes, gene expression of PFF-induced Wnt target genes was suppressed by the Wnt antagonist sFRP4, suggesting that loading activates the Wnt canonical pathway through functional Wnt production. The NO inhibitor L-NAME suppressed the effect of PFF on gene expression of Wnt target genes, suggesting that NO might play a role in PFF-induced Wnt production. The response to PFF differed in MC3T3-E1 osteoblasts."

"The Wnt canonical pathway is activated when a Wnt molecule binds to the cell surface receptor complexes consisting of human lowdensity lipoprotein (LDL) receptor-related protein 5/6 (LRP5/6) and frizzled transmembrane proteins (Fzd)"<-This leads to increased Beta-catenin levels.

"Messenger RNA expression of Wnt3a and the Wnt antagonist SFRP4 was observed in both osteocytes and osteoblasts. Wnt5a gene expression was quantifiable in MC3T3-E1 osteoblasts, but not in MLO-Y4 osteocytes."

"PFF significantly increased NO production at 5 min in MLO-Y4 osteocytes by 4.6-fold, and in MC3T3-E1 osteoblasts by 3.4-fold. The stimulatory effect continued up to 60 min in both cell types"

Voltage profile generation for simultaneous multi-protein detection in western blot analysis

"Protein samples were harvested from MC3T3 osteoblast like cells. Cells were cultured on collagen coated glass slides in MEM medium containing 10% fetal bovine serum and antibiotics. At 80% confluence, cells were serum starved for 12 h, and they were subjected to uni- form flow shear stress at 10 dyn/cm2 for 1 h"

Akt signaling increased in osteoblasts between 0-40 minutes of fluid shear stress.  Flow dramatically increased p-38, p-ERK, and slightly increased p-Akt.  Thus some of the increase in ERK-p and p-38 detection in LSJL may be due to it's stimulation in osteoblasts but not so much for Akt.

Differential Activation and Inhibition of RhoA by Fluid Flow Induced Shear Stress in Chondrocytes.

"Activities of GTPase RhoA in chondrocytes are dependent on intensities of flow induced shear stress. RhoA activities can be either elevated or reduced by selecting different levels of shear stress intensities. C28/I2 chondrocytes have increased RhoA activities in response to high shear stress (10 or 20 dyn/cm(2) ), whereas a decrease in activity was seen with an intermediate shear stress of 5 dyn/cm(2). No changes were seen under low shear stress (2 dyn/cm(2) ). The observed 2-level switch of RhoA activities is closely linked to the shear stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. In chondrocytes, expression of various metalloproteinases is, in part, regulated by shear and normal stresses through a network of GTPases."

"Moderate mechanical loading, for instance, is reported to decrease proteolytic activities of degenerative enzymes in the articular cartilage, while excessive loading may lead to an increase in expression of matrix metalloproteinases"<-This isn't necessarily a bad thing as some MMP's like MMP13 are associated with endochondral ossification.

"Rho-associated kinase (ROCK), promotes the assembly of actin cytoskeleton and phosphorylation of myosin light chains. By regulating intracellular tension through the cytoskeleton, this RhoA-ROCK signaling alters cell shape, and migration patterns as well as cellular differentiation"

"To test the role of intracellular tension in shear stress-induced RhoA activity, we used ML-7 to inhibit myosin light chain kinase or blebbistatin (Bleb) to inhibit non-muscle myosin II. Pretreating with ML-7 (25 μM) or Bleb (50 μM) also prevented shear stress-induced RhoA activation and inhibition at corresponding shear stress levels"

"myosin II-dependent, tensed actin cytoskeleton is necessary for selective RhoA regulation by shear stress regardless of the shear stress magnitude."

"Chondrocytes are in general rich in cortical actin but poor in cytosolic stress fibers. When they dedifferentiate to fibroblast-like cells, they are reported to develop stress fibers. Shapes and differentiation states of chondrocytes are regulated differentially by intermediate and high shear stresses"

Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts.

"Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified."

" β-catenin in the cytoplasm is stabilized by the inactivation of a destruction complex such as axin and GSK3β (glycogen synthase kinase 3β) and translocated to the nucleus. The β-catenin in the nucleus associates with TCF/LEF (T-cell factor/lymphocyte enhancing factor) transcription factors, leading to the activation of TCF/LEF and induction of expression of Wnt target genes"

That was a Yokota study.  So Beta-Catenin is likely stabilized in the osteoblasts by LSJL.  How that can apply to height increase is unclear.

Another Yokota study:

RhoA GTPase interacts with beta-catenin signaling in clinorotated osteoblasts.

"Does unloading suppress an activation level of RhoA GTPase and β-catenin signaling in osteoblasts? If yes, what is the role of RhoA GTPase and actin filaments in osteoblasts in regulating β-catenin signaling? Using a fluorescence resonance energy transfer (FRET) technique with a biosensor for RhoA together with a fluorescent T cell factor/lymphoid enhancer factor (TCF/LEF) reporter, we examined the effects of clinostat-driven[microgravity simulator] simulated unloading. Both RhoA activity and TCF/LEF activity were downregulated by unloading. Reduction in RhoA activity was correlated to a decrease in cytoskeletal organization of actin filaments. Inhibition of β-catenin signaling blocked unloading-induced RhoA suppression, and dominant negative RhoA inhibited TCF/LEF suppression. On the other hand, a constitutively active RhoA enhanced unloading-induced reduction of TCF/LEF activity. The TCF/LEF suppression by unloading was enhanced by co-culture with osteocytes, but it was independent on the organization of actin filaments, myosin II activity, or a myosin light chain kinase. Collectively, the results suggest that β-catenin signaling is required for unloading-driven regulation of RhoA, and RhoA, but not actin cytoskeleton or intracellular tension, mediates the responsiveness of β-catenin signaling to unloading."

"simulated unloading by clinorotation reduces β-catenin signaling activity of MC3T3-E1 cells"

"RhoA is activated by fluid flow and its activation mediates fluid flow-induced PI3K and MAPK signaling"

Fluid flow in the osteocyte mechanical environment: a fluid-structure interaction approach.

"Osteocytes are an elastic cellular structure that deforms in response to the external fluid flow imposed by mechanical loading. The objective of this study is to employ fluid-structure interaction (FSI) modelling to investigate the complex mechanical environment of osteocytes in vivo. By simulating loading levels representative of vigorous physiological activity (3000mupu compression and 300 Pa pressure gradient), we predict average interstitial fluid velocities(60.5mupu/s and average maximum shear stresses  surrounding osteocytes in vivo. Interestingly, these values occur in the canaliculi around the osteocyte cell processes and are within the range of stimuli known to stimulate osteogenic responses by osteoblastic cells in vitro. The greatest mechanical stimulation of the osteocyte occurs in the cell processes, the most mechanosensitive area of the cell."

"Loading the bone matrix surrounding osteocytes generates a pressure differential that drives flow of interstitial fluid within the lacunar–canalicular network. The fluid flow generates a shear stress on the osteocyte cell membrane"

"pressure gradients within individual canaliculi [can be] as high as 1 Pa/nm, the equivalent of an approximately 800 Pa pressure gradient along the length of a single canaliculus"

"within the range of 0.1–2.2 Pa, which has been shown in cell culture studies of osteoblastic cells to result in increased nitric oxide (NO), prostaglandin ( PGE 2) and osteopontin production"<-These chemicals can affect MSCs and chondrocytes.  Calcium signaling also increases which too can impact MSCs.

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