Search

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.

Mechanical strain, testosterone, and estrogen all stimulate proliferation of primary cultures of male rat long bone (LOB)-derived osteoblast-like cells as determined by [3H]thymidine incorporation. The maximum proliferative effect of a single period of mechanical strain (3400 microepsilon, 1 Hz, and 600 cycles) is additional to that of testosterone (10(-8) M) or estrogen (10(-8) M). The cells' proliferative response to strain is abolished both by concentrations of tamoxifen that cause proliferation (10(-8) M) and by those that have no effect (10(-6) M). Strain-related proliferation also is reduced by the estrogen antagonist ICI 182,780 (10(-8) M) but is unaffected by the androgen receptor antagonist hydroxyflutamide (10(-7) M). Tamoxifen, ICI 182,780, and the aromatase inhibitor 4-dihydroandrostenedione, at concentrations that have no effect on basal proliferation, significantly reduce the proliferative effect of the aromatizable androgen testosterone but not that of the nonaromatizable androgen 5alpha-dihydrotestosterone. Hydroxyflutamide, at a concentration that has no effect on basal proliferation (10(-7) M), eliminates the proliferative effect of 5alpha-dihydro-testosterone but had no significant effect on that caused by testosterone. Proliferation associated with strain is blocked by neutralizing antibody to insulin-like growth factor II (IGF-II) but not by antibody to IGF-I. Proliferation associated with testosterone is blocked by neutralizing antibody to IGF-I but is unaffected by antibody to IGF-II. These data suggest that in rat osteoblast-like cells from males, as from females, strain-related proliferation is mediated through the estrogen receptor (ER) in a manner that does not compete with estrogen but that can be blocked by ER modulators. Proliferation associated with testosterone appears to follow its aromatization to estrogen and is mediated through the ER, whereas proliferation associated with 5alpha-dihydrotestosterone is mediated by the androgen receptor. Strain-related proliferation in males, as in females, is mediated by IGF-II, whereas proliferation associated with estrogen and testosterone is mediated by IGF-I.

The involvement of the estrogen receptor in the early responses of bone cells to mechanical strain was investigated by subjecting subconfluent monolayer cultures of ROS.SMER #14 cells (ROS 17/2.8 cells stably transfected with additional ER alpha) to 17 beta-estradiol or a single short period of dynamic mechanical strain (600 cycles, 1 Hz). The basal proliferation rate of ROS.SMER #14 cells was similar to ROS 17/2.8 cells, whose proliferative responsiveness to strain and estrogen is similar to that of primary cultures of rat long bone-derived osteoblasts. At peak strains of 3400 mu epsilon, strain-related proliferation in ROS.SMER #14 cells was 1.4 times that of ROS 17/2.8 cells. At 10(-8) mol/L, 17 beta-estradiol-related proliferation was nearly twice greater. The ROS.SMER #14 cells were transiently transfected with an estrogen-responsive reporter, 2ERE-pS2-CAT, containing two consensus estrogen response elements (ERE) linked to a chloroamphenicol acetyl transferase gene. Strain increased normalized ERE-CAT activity threefold and estradiol (10(-8) mol/L) sixfold. Both strain-related and estradiol-related increases in proliferation and ERE-CAT activity were blocked by the estrogen antagonist ICI 182,780 (10(-6) mol/L). These data show that strain as well as estrogen stimulates increased proliferation in ROS 17/2.8 cells and increased ER alpha-related ERE activity in ROS cells transfected with ER alpha. Proliferation is greater in the cells with more estrogen receptors. Both strain- and estrogen-related proliferation and ERE activity are blocked by the estrogen antagonist ICI 182,780. This indicates that ROS cells' early responses to mechanical strain involve ER alpha and estrogen-responsive genes.

The mechanism by which mechanical strain stimulates bone cell proliferation was investigated and compared with that of estrogen in ROS 17/2.8 cells. Similarity of strain-related responses between ROS cells and osteoblasts was established by demonstrating that ROS cells respond to a short single period of strain in their substrate (1000-3500 microepsilon, 600 cycles, 1 Hz) by a similar strain magnitude-related increase in glucose 6-phosphate dehydrogenase activity as rat osteoblasts and osteocytes in explants in situ. ROS17/2.8 cells also showed similar proliferative responses to strain and 17beta-estradiol, as assessed by [3H]thymidine incorporation and cell counting, as primary cultures of long bone-derived osteoblast-like cells. Strain-related increase in proliferation in ROS cells was accompanied by a 4-fold increase in levels of insulin-like growth factor-II (IGF-II) in conditioned medium. Neither strain nor estrogen had an effect on the conditioned medium levels of IGF-I. Exogenous truncated IGFs tIGF-I and tIGF-II both increased proliferation in a dose-dependent manner. The neutralizing monoclonal antibody (nMAb) to IGF-I blocked proliferation stimulated by tIGF-I but not that due to tIGF-II and vice versa. IGF-I receptor blocking antibody (IGF-IRBAb) blocked the proliferative effect of tIGF-I but not that to tIGF-II. The proliferative effect of estrogen was abolished by IGF-I nMAb and IGF-IRBAb, but these antibodies had no effect on the proliferative response to strain. In contrast IGF-II nMAb abolished the proliferative effect of strain but had no effect on that of estrogen. These data show that ROS17/2.8 cells have similar responses to strain and estrogen qualitatively and quantitatively as rat osteoblasts in situ and rat long bone-derived osteoblast-like cells in primary culture. Estrogen-related proliferation in ROS17/2.8 cells appears to be mediated by IGF-I acting through the IGF-I receptor and does not involve IGF-II. In contrast, strain-related proliferation appears to be mediated by IGF-II and does not involve either IGF-I or the IGF-I receptor.

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.

The estrogen receptor's role in bone cells' response to mechanical strain was investigated by studying the effect of the estrogen receptor modulators ICI 182, 780 and tamoxifen on the proliferation of primary cultures of rat long bone-derived osteoblasts stimulated by the independent and combined effects of 17beta-estradiol, mechanical strain, and the mitogens basic fibroblast growth factor (bFGF), truncated insulin-like growth factor (tIGF)-I and tIGF-II, and epidermal growth factor (EGF). 17Beta-estradiol (10(-10) M to 10(-8) M) increased [3H]thymidine incorporation equally in cells from males and females, as did a single period of cyclical strain in the plastic strips onto which the cells had been seeded (peak strain 3,400 microepsilon, 600 cycles, 1 Hz). At 10(-8) M, neither ICI 182,780 nor tamoxifen had any effect on basal [3H]thymidine incorporation in these cells, but both compounds prevented their proliferative responses to 10(-8) M 17beta-estradiol. Tamoxifen eliminated and ICI 182,780 substantially reduced the proliferation stimulated by strain. 17Beta-estradiol partially rescued the strain-related response from the effect of tamoxifen but not that of ICI 182,780. Both tamoxifen and ICI 182,780 reduced proliferation stimulated by 10(-8) M EGF but had no effect on that by 10(-7) M bFGF or tIGF-I and tIGF-II. That both ICI 182,780 and tamoxifen, which in other tissues act as estrogen antagonists, should reduce osteoblast proliferation stimulated by 17beta-estradiol and EGF, but not that by FGF or the IGFs, was expected since the mitogenic effects of estrogen and EGF involve the estrogen receptor, whereas those of FGF and the IGFs do not. That these compounds should prevent osteoblasts' proliferative response to strain suggests that strain also stimulates mitogenesis by a mechanism involving the estrogen receptor. If this is so, bones' reduced ability to maintain their structural strength after the menopause could be explained by less effective strain-related (re)modeling when estrogen is absent and, among other changes, the estrogen receptor could be down-regulated.

A number of early biochemical responses of bone cells to mechanical loading have been identified, but the full sequence of events from the sensing of strain to the formation of new bone is poorly characterized. Extracellular matrix proteins can modulate cell behavior and would be ideal molecules to amplify the early response to loading. The extracellular matrix protein, tenascin-C, supports differentiation of cultured osteoblast-like cells. The current study was carried out to investigate whether expression patterns of tenascin-C in loaded bones support a role for this protein as a mediator of the osteoregulatory response to loading. Tenascin-C expression was investigated by Northern blot analysis in rat ulnae subjected to an established noninvasive loading regimen engendering physiological strain levels. RNA extracted from loaded compared with contralateral control bones 6 h after loading showed a significant increase in tenascin-C transcript expression. The presence of tenascin-C was investigated by immunohistochemistry in bones of animals killed 3, 5, or 15 days after the initiation of daily loading. In animals killed at 3 or 5 days, periosteal surfaces undergoing load-induced reversal from resorption to formation showed enhanced tenascin-C staining. In animals killed at 15 days, the bone formed in response to loading was clearly demarcated from old bone by strong tenascin-C staining of reversal lines. Within this new bone, tenascin-C staining was seen in the lacunae of older but not more recently embedded osteocytes. The results presented here indicate that tenascin-C expression by bone cells is enhanced in the early osteogenic response to loading. This may indicate that tenascin-C acts as a mediator of the mechanically adaptive response.

The involvement of functional ion channels in previously documented early responses of osteocytes and osteoblasts to mechanical strain in bone tissue was investigated in explants of rat ulnae by the use of ion channel blockers. Gadolinium chloride (a blocker of stretch/shear-sensitive cation channels) elevated basal prostaglandin (PG) E2 and prostacyclin (PGI2) release and osteocyte glucose-6-phosphate dehydrogenase (G6PD) activity, but was associated with a reduction in basal nitric oxide (NO) production. Gadolinium abolished loading-related increases in the release of PGI2 and NO and osteocyte G6PD activity. Gadolinium also reduced the loading-related release of PGE2 assumed to originate from osteoblasts and the magnitude of loading-related increases in G6PD activity in these cells. Nifedipine (a blocker of L-type voltage-dependent calcium channels) had no effect on basal levels of prostanoid or NO release, or G6PD activity in osteocytes or osteoblasts, and did not affect loading-related release of PGI2 or increase in osteocyte G6PD. However, nifedipine prevented loading-related increases in PGE2 and NO release and osteoblast G6PD activity. These results are consistent with osteocytes' response to bone loading requiring activatable ion channels sensitive to gadolinium, but not those sensitive to nifedipine. In osteoblasts, the early responses to bone loading appear to be associated with ion channels sensitive to gadolinium and nifedipine; however, the nifedipine-sensitive channels seem to have the dominant effect.

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 stimulated 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.

The tibiae and fibulae of 14-week-old rats were subjected to a single 5 minutes period of cyclic longitudinal loading at 1 Hz. The activity of the enzymes glucose 6-phosphate dehydrogenase (G6PD) and alkaline phosphatase (ALP) in osteocytes and periosteal osteoblasts was measured immediately and 24 h after loading. In osteocytes G6PD activity was increased immediately after loading but returned to control values 24 h later. There was no detectable ALP activity in these cells regardless of loading history. In periosteal osteoblasts G6PD activity was raised immediately after loading and remained higher than controls 24 h later. ALP activity in periosteal cells was unaffected immediately after loading but 24 h later was substantially increased. These findings are consistent with osteocytes and periosteal cells both being immediately responsive to periods of intermittent loading in their adjacent matrices. In both cell types an early feature of this response is an increase in G6PD activity. In osteocytes this response is short-lived, suggesting that it is an early biochemical change associated with strain perception that does not progress to matrix synthesis. The increase in G6PD activity with unaffected ALP levels in periosteal cells immediately after loading is consistent with a similar response. In these cells the increase in G6PD accompanied by increased ALP levels 24 h after loading suggests that the loading-related response progresses to new bone formation.

Pairs of 17-day embryonic chick tibiotarsi were removed and maintained in organ culture. One of each pair was subjected to a single 20-minute period of intermittent loading at 0.4 Hz, producing peak longitudinal compressive strains of 650 microstrain (mu epsilon). In the 18-hour culture period following loading, alkaline phosphatase levels in the osteoblasts of the loaded tibiotarsi were maintained whereas in controls they declined. In situ hybridization using a collagen type I cRNA riboprobe showed a substantial increase in expression of mRNA for collagen type I in the periosteal tissue of bones that were cultured for 18 hours after loading compared with that in similarly cultured controls and bones cultured for 4 hours. These results demonstrate that appropriate loading of embryonic chick bones in organ culture elicits adaptive regulation of matrix synthesis as evidenced by increased expression of the gene for type I collagen and alkaline phosphatase activity. This model may be useful as it must contain all the obligatory steps between strain change in the matrix and modified osteogenic activity.