Andrew A. Pitsillides

Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone (re)modeling?

The structural competence of the skeleton is maintained by an adaptive mechanism in which resident bone cells respond to load‐induced strains. To investigate the possible role of the messenger molecule nitric oxide (NO) in this response, we studied NO production in well‐characterized organ culture systems, rat long bone‐derived osteoblast‐like (LOBs) cells, and embryonic chick osteocytes (LOCYs) in monolayer culture. In superfused cancellous bone cores, loading (for 15 min) produces increases in NO2‐ (stable NO metabolite) release during the loading period, which paralleled those in PGI2 and PGE2‐ Loading of rat vertebrae and ulnae produces increases in NO2‐ release, and in ulnae NO synthase inhibitors diminish these responses. Transient rapid increases in NO release are stimidated by strain in both LOBs and LOCYs. Polymerase chain reaction amplification of extracted mRNA shows that rat ulnae, LOBs, and LOCYs express both the inducible and neuronal (constitutive) isoforms of NO synthase. Adaptability to mechanical strain relies on assessment of the strain environment followed by modification of bone architecture. Immediate increases in NO production induced by loading suggest the involvement of NO in strain measurement and cellular communication to establish strain distribution, as well as potentially in adaptive changes in bone cell behavior.—Pitsillides, A. A., Rawlinson, S. C. F., Suswillo, R. F. L., Bourrin, S., Zaman, G., Lanyon, L. E. Mechanical strain‐induced NO production by bone cells: a possible role in adaptive bone (re)modeling? FASEB J. 9, 1614‐1622(1995)

Mechanical Strain Stimulates Nitric Oxide Production by Rapid Activation of Endothelial Nitric Oxide Synthase in Osteocytes

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast‐like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading‐induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non–load‐bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain‐induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast‐like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.

Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signaling pathways

Extracellular regulated kinases (ERKs)-1 and -2 are members of the MAPK family of protein kinases involved in the proliferation, differentiation, and apoptosis of bone cells. We have shown previously that ROS 17/2.8 cells show increased activation of ERK-1 or -2, which is sustained for 24 h, when the strips onto which they are seeded are subjected to a 10 min period of cyclic four point bending that produces physiological levels of mechanical strain along with associated fluid movement of the medium. Movement of the strips through the medium without bending causes fluid movement without strain. This also increases ERK-1/2 activation, but in a biphasic manner over the same time period. Our present study investigates the role of components of signaling pathways in the activation of ERK-1/2 in ROS 17/2.8 cells in response to these stimuli. Using a range of inhibitors we show specific differences by which ERK-1 and ERK-2 are activated in response to fluid movement alone, compared with those induced in response to strain plus its associated fluid movement. ERK-1 activation induced by fluid movement was markedly reduced by nifedipine, and therefore appears to involve L-type calcium channels, but was unaffected by either L-NAME or indomethacin. This suggests independence from prostacyclin (PGI(2)) and nitric oxide (NO) production. In contrast, ERK-1 activation induced by application of strain (and its associated fluid disturbance) was abrogated by TMB-8 hydrochloride, L-NAME, and indomethacin. This suggests that strain-induced ERK-1 activation is dependent upon calcium mobilization from intracellular stores and production of NO and PGI(2). ERK-2 activation appears to be mediated by a separate mechanism in these cells. Its activation by fluid movement alone involved both PGI(2) and NO production, but its activation by strain was not affected by any of the inhibitors used. The G protein inhibitor, pertussis toxin, did not cause a reduction in the activation of ERK-1 or -2 in response to either stimulus. These results are consistent with earlier observations of ERK activation in bone cells in response to both strain (with fluid movement) and fluid movement alone, and further demonstrate that these phenomena stimulate distinct signaling pathways.

Hyaluronan synthesis and degradation in cartilage and bone

Abstract.Hyaluronan (HA) is a large but simple glycosaminoglycan composed of repeating D-glucuronic acid, β1–3 linked to N-acetyl-D-glucosamine β1–4, found in body fluids and tissues, in both intra- and extracellular compartments. Despite its structural simplicity, HA has diverse functions in skeletal biology. In development, HA-rich matrices facilitate migration and condensation of mesenchymal cells, and HA participates in joint cavity formation and longitudinal bone growth. In adult cartilage, HA binding to aggrecan immobilises aggrecan, retaining it at the high concentrations required for compressive resilience. HA also appears to regulate bone remodelling by controlling osteoclast, osteoblast and osteocyte behaviour. The functions of HA depend on its intrinsic properties, which in turn rely on the degree of polymerisation by HA synthases, depolymerisation by hyaluronidases, and interactions with HA-binding proteins. HA synthesis and degradation are closely regulated in skeletal tissues and aberrant synthetic or degradative activity causes disease. The role and regulation of HA synthesis and degradation in cartilage, bone and skeletal development is discussed.

Osteocytes Use Estrogen Receptor α to Respond to Strain but Their ERα Content Is Regulated by Estrogen

The role of mechanical strain and estrogen status in regulating ERα levels in bone cells was studied in female rats. OVX is associated with decreased ERα protein expression/osteocyte, whereas habitual strain and artificial loading has only a small but positive effect, except on the ulnas medial surface, where artificial loading stimulates reversal of resorption to formation.

An essential role for the interaction between hyaluronan and hyaluronan binding proteins during joint development.

We studied the expression of hyaluronan binding proteins (HABPs) during the development of embryonic chick joints, using immunocytochemistry and biotinylated HA. The expression of actin capping proteins and of actin itself was also studied because the cytoskeleton is important in controlling HA-HABP interactions. Three cell surface HABPs were localized in the epiphyseal cartilage, articular fibrocartilage, and interzone that comprise the developing joint. Of these three HABPs, CD44 was associated with the articular fibrocartilages and interzone, whereas RHAMM and the IVd4 epitope were associated with all three tissues. Biotinylated HA was localized to interzone and articular fibrocartilages before cavity formation and within epiphyseal chondrocytes post cavitation. Actin filament bundles were observed at the developing joint line, as was the expression of the actin capping protein moesin. Manipulation of joint cavity development, using oligosaccharides of HA, disrupted joint formation and was associated with decreases in CD44 and actin filament expression as well as decreased hyaluronan synthetic capability. These results suggest that HA is actively bound by CD44 at the developing joint line and that HA-HABP interactions play a major role in the initial separation events occurring during joint formation.

Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells†

Communication between endothelial and bone cells is crucial for controlling vascular supply during bone growth, remodeling, and repair but the molecular mechanisms coordinating this intercellular crosstalk remain ill‐defined. We have used primary human and rat long bone‐derived osteoblast‐like cells (HOB and LOB) and human umbilical vein endothelial cells (HUVEC) to interrogate the potential autocrine/paracrine role of vascular endothelial cell growth factor (VEGF) in osteoblast:endothelial cell (OB:EC) communication and examined whether prostaglandins (PG), known modulators of both OB and EC behavior, modify VEGF production. We found that the stable metabolite of PGI2, 6‐keto‐PGF1α and PGE2, induced a concentration‐dependent increase in VEGF release by HOBs but not ECs. In ECs, VEGF promoted early ERK1/2 activation, late cyclooxygenase‐2 (COX‐2) protein induction, and release of 6‐keto‐PGF1α. In marked contrast, no significant modulation of these events was observed in HOBs exposed to VEGF, but LOBs clearly exhibited COX‐dependent prostanoid release (10‐fold less than EC) following VEGF treatment. A low level of osteoblast‐like cell responsiveness to exogenous VEGF was supported by VEGFR2/Flk‐1 immunolabelling and by blockade of VEGF‐mediated prostanoid generation by a VEGFR tyrosine kinase inhibitor (TKI). HOB alkaline phosphatase (ALP) activity was increased following long‐term non‐contact co‐culture with ECs and exposure of ECs to VEGF in this system further increased OB‐like cell differentiation and markedly enhanced prostanoid release. Our studies confirm a paracrine EC‐mediated effect of VEGF on OB‐like cell behavior and are the first supporting a model in which prostanoids may facilitate this unidirectional VEGF‐driven OB:EC communication. These findings may offer novel regimes for modulating pathological bone remodeling anomalies through the control of the closely coupled vascular supply. J. Cell. Physiol. 214: 537–544, 2008.

Cartilage biology in osteoarthritis—lessons from developmental biology

The cellular and molecular mechanisms responsible for the initiation and progression of osteoarthritis (OA), and in particular cartilage degeneration in OA, are not completely understood. Increasing evidence implicates developmental processes in OA etiology and pathogenesis. Herein, we review this evidence. We first examine subtle changes in cartilage development and the specification and formation of joints, which predispose to OA development, and second, we review the switch from an articular to a hypertrophic chondrocyte phenotype that is thought to be part of the OA pathological process ultimately resulting in cartilage degeneration. The latest studies are summarized and we discuss the concepts emerging from these findings in cartilage biology, in the light of our understanding of the developmental processes involved.

Ammonia impairs neutrophil phagocytic function in liver disease

Hyperammonemia is a feature of liver failure, which is associated with increased risk of infection. The aims of the present study were to determine in vitro, in rats fed an ammoniagenic diet and in patients with cirrhosis, whether induction of hyperammonemia results in neutrophil dysfunction. As hyperammonemia produces cell swelling, we explored the role of the osmoregulating, p38 mitogen‐activated protein kinase (p38MAPK) pathway in mediating this neutrophil dysfunction. Neutrophils were isolated from blood of healthy volunteers and incubated with either 75 μM ammonia or phosphate‐buffered saline. Both groups were studied under hyponatremic conditions and/or with the addition of p38MAPK modulators. Neutrophil phagocytosis was measured in naive rats and rats fed an ammoniagenic diet and in patients with stable cirrhosis given placebo (n = 8) or an amino acid solution inducing hyperammonemia (n = 8). Cell volume and phagocytosis was analyzed by fluorescent‐activated cell sorting using fluorescein isothiocyanate–labeled E. coli. p38MAPK phosphorylation was measured by western blotting. In healthy neutrophils incubated with ammonia and in rats fed an ammoniagenic diet, neutrophils showed evidence of swelling, impaired phagocytosis, and increased spontaneous oxidative burst compared to controls. Phagocytosis was significantly impaired in patients with induced hyperammonemia compared to placebo. The effects of hyperammonemia and hyponatremia were synergistic. The p38MAPK intracellular signaling pathways were activated in healthy neutrophils exposed to ammonia in association with increased burst activity. Neutrophil phagocytic dysfunction was abrogated by the addition of a p38MAPK agonist. Conclusion: Ammonia produces neutrophil swelling and impairs neutrophil phagocytosis. The p38MAPK intracellular signaling pathway has been shown to be important in mediating the ammonia‐induced neutrophil dysfunction. (HEPATOLOGY 2008.)

Human Osteoblasts' Proliferative Responses to Strain and 17β-Estradiol Are Mediated by the Estrogen Receptor and the Receptor for Insulin-Like Growth Factor I†

The mechanism by which mechanical strain and estrogen stimulate bone cell proliferation was investigated using monolayer cultures of human osteoblastic TE85 cells and female human primary (first‐passage) osteoblasts (fHOBs). Both cell types showed small but statistically significant dose‐dependent increases in [3H]thymidine incorporation in response to 17β‐estradiol and to a single 10‐minute period of uniaxial cyclic strain (1 Hz). In both cell types, the peak response to 17β‐estradiol occurred at 10−8‐10−7 M and the peak response to strain occurred at 3500 microstrain (μϵ). Both strain‐related and 17β‐estradiol‐related increases in [3H]thymidine incorporation were abolished by the estrogen receptor (ER) modulator ICI 182,780 (10−8 M). Tamoxifen (10−9‐10−8 M) increased [3H]thymidine incorporation in both cell types but had no effect on their response to strain. In TE85 cells, tamoxifen reduced the increase in [3H]thymidine incorporation associated with 17β‐estradiol to that of tamoxifen alone but had no such effect in fHOBs. In TE85 cells, strain increased medium concentrations of insulin‐like growth factor (IGF) II but not IGF‐I, whereas 17β‐estradiol increased medium concentrations of IGF‐I but not IGF‐II. Neutralizing monoclonal antibody (MNAb) to IGF‐I (3 μg/ml) blocked the effects of 17β‐estradiol and exogenous truncated IGF‐I (tIGF‐I; 50 ng/ml) but not those of strain or tIGF‐II (50 ng/ml). Neutralizing antibody to IGF‐II (3 μg/ml) blocked the effects of strain and tIGF‐II but not those of 17β‐estradiol or tIGF‐I. MAb αIR‐3 (100 ng/ml) to the IGF‐I receptor blocked the effects on [3H]thymidine incorporation of strain, tIGF‐II, 17β‐estradiol, and tIGF‐I. HOBs and TE85 cells, act similarly to rat primary osteoblasts and ROS 17/2.8 cells in their dose‐related proliferative responses to strain and 17β‐estradiol, both of which can be blocked by the ER modulator ICI 182,780. In TE85 cells (as in rat primaries and ROS 17/2.8 cells), the response to 17β‐estradiol is mediated by IGF‐I, and the response to strain is mediated by IGF‐II. Human cells differ from rat cells in that tamoxifen does not block their response to strain and reduces the response to 17β‐estradiol in TE85s but not primaries. In both human cell types (unlike rat cells) the effects of strain and IGF‐II as well as estradiol and IGF‐I can be blocked at the IGF‐I receptor.