Stretch-induced phosphorylation of ERK1/2 depends on differentiation stage of osteoblasts.

The goal of this study was to investigate the effect of mechanical loading on osteoblasts and extracellular signal-regulated kinase (ERK1/2) signaling in relation to osteoblast differentiation and mineralization. A human osteoblast cell line (SV-HFO) was triggered to differentiate to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and beta-glycerophosphate. Osteoblasts were subjected to cyclic, equibiaxial stretch for 5, 15, or 60 min at different stages of differentiation (days 7, 14, and 21). Baseline (static) phosphorylated ERK1/2 and total ERK1/2 levels gradually increased during osteoblast differentiation. Cyclic stretch induced a rapid increase in ERK1/2 phosphorylation with a maximum between 5 and 15 min. Prolonged stretching for 60 min resulted in a decrease of phosphorylated ERK1/2 towards baseline level, suggesting a desensitization mechanism. The effect of stretch on ERK1/2 phosphorylation was strongest at later stages of differentiation (days 14 and 21). At day 21, the increase of ERK1/2 phosphorylation in response to stretch was significantly lower in non-differentiating than in differentiating osteoblasts. This could not be explained by differences in cell density, but did correlate with the formation of extracellular matrix, collagen fibrils. Mineralization of the extracellular matrix did not lead to a further increase of ERK1/2 phosphorylation. In conclusion, the current study demonstrates that the extent of activation of the ERK1/2 pathway is dependent on the differentiation or functional stage of the osteoblast. The presence of an extracellular matrix, but not per se mineralization, seems to be the predominant determinant of osteoblastic response to strain.

Mechanical control of human osteoblast apoptosis and proliferation in relation to differentiation.

Bone cells respond to mechanical stimulation. This is thought to be the mechanism by which bone adapts to mechanical loading. Reported responses of bone cells to mechanical stimuli vary widely and therefore there is no consensus on what mechanisms of mechanotransduction are physiologically relevant. We hypothesize that the differentiation stage of osteoblastic cells used to study responses to strain in vitro determines the outcome of applied loading. A human fetal osteoblast cell line was triggered to differentiate in culture to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and b-glycerophosphate. Osteoblast cultures were subjected to increasing levels of cyclic, equibiaxial stretch at different stages of differentiation. We show that differentiation of human osteoblasts affects their responses to stretch in vitro. In 7-day osteoblast cultures, stretch results in decreased cell numbers as cells are triggered into apoptosis, independent of the stretch level (between 0.4-2.5%). In more mature cultures, apoptosis is not affected by the same treatment. Stretching differentiating cultures at day 14 actually increases proliferation. This is the first study reporting on differentiation-dependent mechanical control of osteoblast proliferation and apoptosis and is fundamental in understanding mechanotransduction processes in bone. The tight regulation of these responses by differentiation implies the significance of the differentiation stage of osteoblasts for the translation of mechanical signals and corroborates with the putative role of the osteoblastic lineage as mechanotransducer in bone.

ERK activation and alpha v beta 3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts.

Osteoblast growth and differentiation are critical processes for bone development and maintenance, and are regulated by both humoral and mechanical factors. Humoral (hormonal) factors can affect gene transcription via MAPkinases, e.g., extracellular signal-regulated kinase (ERK). We studied whether the ERK pathway is also involved in processing mechanical inputs in human bone cells. Exposing MG63 cells to physiologically relevant levels of fluid flow resulted in ERK phosphorylation. Genistein blocked this response, indicating that it is dependent on tyrosine phosphorylation. Furthermore, alpha v beta 3 integrins were activated in response to fluid flow, as shown by recruitment of adaptor molecule Shc and clustering of alpha v beta 3 in focal adhesion-like structures. Antibodies blocking formation of beta 1 or beta 3 integrin-matrix interactions or RGD peptides could not inhibit fluid flow-induced ERK phosphorylation, suggesting that formation of new integrin-matrix interactions is not essential for this response and that other upstream mechanosensors regulate induction of ERK phosphorylation in response to fluid flow in human bone cells.

Characterisation of glucocorticoid receptors in peripheral blood leukocytes of Carp, Cyprinus carpio L.

Binding studies with [3H]cortisol revealed the presence of a single class of cortisol-binding sites on carp peripheral blood leukocytes (PBL). These binding sites showed high affinity (Kd of 3.8 nM) and low capacity (490 binding sites per cell), indicative of receptor binding. Affinity for cortisone was 254-fold lower than for cortisol. Affinity for the two synthetic glucocorticoids dexamethasone and triamcinolone acetonide (TA) was 4- and 10-fold higher than for cortisol, respectively. Further evidence for the GR character of the receptor came from results showing that cortisol induced apoptosis, which could be blocked by the glucocorticoid analogue RU486. A single meal of cortisol-containing food elevated plasma cortisol concentrations and decreased GR density in PBL, as measured 3 h later. The percentage of circulating B lymphocytes also decreased. Cortisol-induced redistribution of B lymphocytes from the blood, due to cortisol treatment, may explain the decrease of GR numbers in PBL, although downregulation of available GR remains possible.

Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of the common carp, Cyprinus carpio L..

In mammalian T and B cells glucocorticoids (GS) regulate development and selection through induction of apoptosis; more recently GS-induced apoptosis has also been implicated in the removal of circulating, activated T and B cells following an immune response. In an earlier report we have given the first evidence for cortisol-induced apoptosis as an immune regulator in an aquatic vertebrate, the common carp. Here we report on subpopulation-specific sensitivity of carp peripheral blood leukocytes (PBL) to cortisol-induced apoptosis. B cells, the most abundant leukocyte subpopulation in fish blood, are sensitised to cortisol-induced apoptosis by activation with the mitogens LPS or PHA. Cortisol-induced apoptosis in B cells is receptor mediated as it is blocked by the synthetic GS receptor blocker RU486. In contrast to what is known for mammalian lymphocytes, apoptosis in carp T cells is hardly affected by cortisol, both in unstimulated and in PHA-stimulated cell cultures. A culture supernatant of PHA-prestimulated PBL, containing IL-2-like activity, decreased spontaneous apoptosis in both T and B cells, but did not affect cortisol-induced apoptosis in B cells. Apoptosis in thrombocytes was unaffected by either mitogens, cortisol, or lymphocyte supernatant. The difference between mammalian and fish leukocyte sensitivity to cortisol is discussed in the light of differences in the immune response of mammals and fish.

Cortisol inhibits apoptosis in carp neutrophilic granulocytes.

The direct effect of cortisol treatment on carp neutrophil viability was examined in vitro. Cortisol treatment caused an inhibition of neutrophil apoptosis. The effect was blocked by glucocorticoid receptor blocker RU486, showing that rescue from apoptosis was receptor mediated. Using binding studies with radioactive cortisol, a single class of glucocorticoid receptors was detected with high affinity (Kd = 2.6 nM) and low capacity (497 receptors/cell) for cortisol binding. Both in vitro and in vivo cortisol treatment did not affect neutrophil respiratory burst activity. These data indicate that cortisol can augment the supply of functional neutrophilic granulocytes in conditions of acute stress, which may be essential for survival, since phagocytes form the first line of defence against micro-organisms.

Conservation of apoptosis as an immune regulatory mechanism: effects of cortisol and cortisone on carp lymphocytes.

This is the first study to show that apoptosis as an immune regulatory mechanism is conserved in fish, demonstrating its importance in maintaining immunological homeostasis. The data further show that this mechanism is subject to control by glucocorticosteroids. Carp plasma cortisol concentrations increase from 20 to 434 ng/ml and cortisone from 5 to 50 ng/ml within 9 min of the onset of handling stress. At basal steroid concentrations in vitro, cortisol, but not its conversion product cortisone, inhibits proliferation of peripheral blood lymphocytes (PBL), as measured by [3H]thymidine incorporation. Induction of apoptosis in activated PBL is the apparent mechanism of cortisol action. In nonstimulated PBL cultures, apoptosis is induced by neglect (a lack of stimulating signals). Stimulation with LPS or PHA rescues lymphocytes from this type of apoptosis. Stimulated PBL populations, however, are sensitive to cortisol-induced apoptosis. Culture supernatants from activated PBL protect PBL from apoptosis by neglect, probably by supplying a growth signal. These supernatants, however, have no effect on cortisol-induced apoptosis.

Carp macrophages and neutrophilic granulocytes secrete an interleukin-1-like factor.

Carp, Cyprinus carpio L, macrophages and neutrophilic granulocytes obtained from pronephros were cultured. Supernatant was harvested after 48 h and tested for interleukin-1 (IL-1) bioactivity. A concentration-dependent stimulation of proliferation was found of carp Ig- lymphocytes as well as of the murine IL-1 dependent D10 (N4) M cell line. A 4 h treatment of cells with phorbol myristate acetate prior to culture gave a two- to fourfold enhancement of the bioactivity in the supernatant. Antibodies raised in sheep against human recombinant IL-1 alpha or IL-1 beta added to the supernatant annulled the IL-1 bioactivity. Western blot analysis of supernatants with sheep or rabbit polyclonal antisera against human IL-1s revealed 22 kDa and 15 kDa protein species. The predominant newly synthesized protein that was immunoprecipitated with these antisera was a 15 kDa molecular species. We conclude that carp macrophages and neutrophilic granulocytes produce an IL-1-like molecule with T-cell proliferating potency that shares structural similarities with mammalian IL-1. This is the first evidence for the IL-1 signal protein in carp immunocompetent cells.