Histochemical analysis reveals organ-specific quantitative trait loci for enzyme activities in Arabidopsis

To identify genetic loci involved in the regulation of organ-specific enzyme activities, a specific histochemical staining protocol was used in combination with quantitative trait locus (QTL) analysis. Using phosphoglucomutase (PGM) as an example, it is shown that enzyme activity can specifically, and with high resolution, be visualized in non-sectioned seedlings of Arabidopsis. The intensities of staining were converted to quantitative data and used as trait for QTL analysis using Landsberg erecta x Cape Verde Islands recombinant inbred lines. Independently, PGM activities were quantified in whole-seedling extracts, and these data were also used for QTL analysis. On the basis of extract data, six significant (P < 0.05) loci affecting PGM activity were found. From the histochemical data, one or more specific QTLs were found for each organ analyzed (cotyledons, shoot apex, hypocotyl, root, root neck, root tip, and root hairs). Loci detected for PGM activity in extracts colocated with loci for histochemical staining. QTLs were found coinciding with positions of (putative) PGM genes but also at other positions, the latter ones supposedly pointing toward regulatory genes. Some of this type of loci were also organ specific. It is concluded that QTL analysis based on histochemical data is feasible and may reveal organ-specific loci involved in the regulation of metabolic pathways.     

Mechanotransduction in bone cells proceeds via activation of COX-2, but not COX-1

Cyclooxygenase (COX) is the key enzyme in the production of prostaglandins, which are essential for the response of bone to mechanical loading. We determined which COX-isoform, COX-1 or COX-2, determines loading-induced prostaglandin production in primary bone cells in vitro. Mouse and human bone cells reacted to 1 h of pulsating fluid flow (PFF, 0.6+/-0.3 Pa at 5 Hz) with an increased prostaglandin E(2) production, which continued 24 h after cessation of PFF. Inhibition of COX-2 activity with NS-398 abolished the stimulating effect of PFF both at 1 h and at 24 h post-incubation, while inhibition of COX-1 by SC-560 affected neither the early nor the late response to flow. PFF rapidly stimulated COX-2 mRNA expression at 1 h but did not affect COX-1 mRNA expression. COX-2 mRNA expression was still significantly enhanced 24 h after cessation of PFF. We conclude that COX-2 is the mechanosensitive form of COX that determines the response of bone tissue to mechanical loading.     

Expression of serotonin receptors in bone

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.     

Extracellular NO signalling from a mechanically stimulated osteocyte

Bone remodelling is a dynamic process that requires the coordinated interaction of osteocytes, osteoblasts, and osteoclasts, collaborating in basic multicellular units (BMUs). Communication between these cells can be by extracellular soluble molecules as well as directly propagating intercellular signalling molecules. Key to the understanding of bone remodelling is osteocyte mechanosensing and chemical signalling to the surrounding cells, since osteocytes are believed to be the mechanosensors of bone, responding to mechanical stresses. Nitric oxide (NO) is an important parameter to study osteocyte activation following mechanical loading. It is a small short-lived molecule, which makes its real-time, quantitative monitoring difficult. However, recently we demonstrated that DAR-4M AM chromophore can be used for real-time quantitative monitoring of intracellular NO production in individual cells following mechanical loading. Here we studied if a single mechanically stimulated osteocyte communicates with, and thus activates its surrounding cells via extracellular soluble factors. We monitored quantitatively intracellular NO production in the stimulated osteocyte and in its surrounding osteocytes, which were not interconnected. Mechanical stimulation by microneedle of a single-MLO-Y4 osteocyte-like cell upregulated the average intracellular NO production by 94% in the stimulated cell, and by 31-150% in the surrounding osteocytes. In conclusion, a single osteocyte can disseminate a mechanical stimulus to its surrounding osteocytes via extracellular soluble signalling factors. This reinforces the putative mechanosensory role of osteocytes, and demonstrates a possible mechanism by which a single mechanically stimulated osteocyte can communicate with other cells in a BMU, which might help to better understand the intricacies of intercellular interactions in BMUs and thus bone remodelling.     

Mechanical Loading by Fluid Shear Stress of Myotube Glycocalyx Stimulates Growth Factor Expression and Nitric Oxide Production

Skeletal muscle fibers have the ability to increase their size in response to a mechanical overload. Finite element modeling data suggest that mechanically loaded muscles in vivo may experience not only tensile strain but also shear stress. However, whether shear stress affects biological pathways involved in muscle fiber size adaptation in response to mechanical loading is unknown. Therefore, our aim was twofold: (1) to determine whether shear stress affects growth factor expression and nitric oxide (NO) production by myotubes, and (2) to explore the mechanism by which shear stress may affect myotubes in vitro. C2C12 myotubes were subjected to a laminar pulsating fluid flow (PFF; mean shear stress 0.4, 0.7 or 1.4 Pa, 1 Hz) or subjected to uni-axial cyclic strain (CS; 15 % strain, 1 Hz) for 1 h. NO production during 1-h PFF or CS treatment was quantified using Griess reagent. The glycocalyx was degraded using hyaluronidase, and stretch-activated ion channels (SACs) were blocked using GdCl₃. Gene expression was analyzed immediately after 1-h PFF (1.4 Pa, 1 Hz) and at 6 h post-PFF treatment. PFF increased IGF-I Ea, MGF, VEGF, IL-6, and COX-2 mRNA, but decreased myostatin mRNA expression. Shear stress enhanced NO production in a dose-dependent manner, while CS induced no quantifiable increase in NO production. Glycocalyx degradation and blocking of SACs ablated the shear stress-stimulated NO production. In conclusion, shear stress activates signaling pathways involved in muscle fiber size adaptation in myotubes, likely via membrane-bound mechanoreceptors. These results suggest that shear stress exerted on myofiber extracellular matrix plays an important role in mechanotransduction in muscle.     

The Effects of Orally Administered Beta-Glucan on Innate Immune Responses in Humans,a Randomized Open-Label Intervention Pilot-Study

Rationale

To prevent or combat infection, increasing the effectiveness of the immune response is highly desirable, especially in case of compromised immune system function. However, immunostimulatory therapies are scarce, expensive, and often have unwanted side-effects. β-glucans have been shown to exert immunostimulatory effects in vitro and in vivo in experimental animal models. Oral β-glucan is inexpensive and well-tolerated, and therefore may represent a promising immunostimulatory compound for human use.

Methods

We performed a randomized open-label intervention pilot-study in 15 healthy male volunteers. Subjects were randomized to either the β -glucan (n = 10) or the control group (n = 5). Subjects in the β-glucan group ingested β-glucan 1000 mg once daily for 7 days. Blood was sampled at various time-points to determine β-glucan serum levels, perform ex vivo stimulation of leukocytes, and analyze microbicidal activity.

Results

β-glucan was barely detectable in serum of volunteers at all time-points. Furthermore, neither cytokine production nor microbicidal activity of leukocytes were affected by orally administered β-glucan.

Conclusion

The present study does not support the use of oral β-glucan to enhance innate immune responses in humans.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01727895","term_id":"NCT01727895"}}NCT01727895     

MT1-MMP modulates the mechanosensitivity of osteocytes

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli. MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP(+/+) and MT1-MMP(-/-) mice. PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP(-/-) bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP(-/-) bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis. In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing.     

Inhibition of osteocyte apoptosis by fluid flow is mediated by nitric oxide

Bone unloading results in osteocyte apoptosis, which attracts osteoclasts leading to bone loss. Loading of bone drives fluid flow over osteocytes which respond by releasing signaling molecules, like nitric oxide (NO), that inhibit osteocyte apoptosis and alter osteoblast and osteoclast activity thereby preventing bone loss. However, which apoptosis-related genes are modulated by loading is unknown. We studied apoptosis-related gene expression in response to pulsating fluid flow (PFF) in osteocytes, osteoblasts, and fibroblasts, and whether this is mediated by loading-induced NO production. PFF (0.7 ± 0.3 Pa, 5 Hz, 1 h) upregulated Bcl-2 and downregulated caspase-3 expression in osteocytes. l-NAME attenuated this effect. In osteocytes PFF did not affect p53 and c-Jun, but l-NAME upregulated c-Jun expression. In osteoblasts and fibroblasts PFF upregulated c-Jun, but not Bcl-2, caspase-3, and p53 expression. This suggests that PFF inhibits osteocyte apoptosis via alterations in Bcl-2 and caspase-3 gene expression, which is at least partially regulated by NO.     

The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes

Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cartilage pathology has been reported. Wnt signaling regulates critical biological processes. Wnt signals are transduced through at least three intracellular signaling pathways including the canonical Wnt/β-catenin pathway, the Wnt/Ca2 + pathway and the Wnt/planar cell polarity pathway. We investigated the involvement of the Wnt canonical and non-canonical pathways in human articular chondrocyte de-differentiation in vitro. Human articular chondrocytes were cultured through four passages with no treatment, or with sFRP3 treatment, an inhibitor of Wnt pathways, or with DKK1 treatment, an inhibitor of the canonical pathway. Chondrocyte-secreted markers and Wnt pathway components were analyzed using western blotting and qPCR. Inhibition of the Wnt pathway showed that the canonical Wnt signaling probably is responsible for inhibition of collagen II expression, activation of metalloproteinase 13 expression and regulation of Wnt7a and c-jun expression during chondrocyte de-differentiation in vitro. Our results also suggest that expressions of eNOS, Wnt5a and cyclinE1 are regulated by non-canonical Wnt signaling.