Mechanical strain and estrogen activate estrogen receptor alpha in bone cells.

Bone cells' early responses to estrogen and mechanical strain were investigated in the ROS 17/2.8 cell line. Immunoblotting with antiphosphorylated estrogen receptor a (ER-alpha) antibody showed that when these cells were exposed for 10 minutes to estrogen (10(-8) M) or a single period of cyclic dynamic strain (peak 3400 microepsilon, 1 Hz, 600 cycles), there was an increase in the intensity of a 66-kDa band, indicating phosphorylation of ser122 in the amino terminus of ER-alpha. Increased phosphorylation was detected within 5 minutes of exposure to estrogen and 5 minutes after the end of the period of strain. Estrogen and strain also activated the mitogen-activated protein kinase (MAPK) family member extracellular regulated kinase-1 (ERK-1). Increases in ERK activation coincided with increased ER-alpha phosphorylation. Activation of ERK-1 and the phosphorylation of ER-alpha, by both estrogen and strain, were prevented by the MAP kinase kinase (MEK) inhibitor U0126 and the protein kinase A (PKA) inhibitor (PKI). These data support previous suggestions that resident bone cells' early responses to strain and estrogen share a common pathway, which involves ER-alpha. This pathway also appears to involve PKA and ERK-mediated phosphorylation of ser122 within the amino terminus of ER-alpha. Reduced availability of this pathway when estrogen levels are reduced could explain diminished effectiveness of mechanically related control of bone architecture after the menopause.

Mechanical strain activates estrogen response elements in bone cells.

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.

Mechanical strain stimulates ROS cell proliferation through IGF-II and estrogen through IGF-I.

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.

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.

Enhancement by sex hormones of the osteoregulatory effects of mechanical loading and prostaglandins in explants of rat ulnae.

Explants of ulnae from 5-week-old male and female rats were cleaned of marrow and soft tissue and, in the presence and absence of 10(-8) M 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT), mechanically loaded or treated with exogenous prostanoids previously shown to be produced during loading. Over an 18-h period, mechanical loading (peak strain 1300 mu epsilon, 1 Hz, 8 minutes, maximum strain rate 25,000 mu epsilon/s), prostaglandin E2 (PGE2) and prostacyclin (PGI2) (10(-6) M), each separately produced quantitatively similar increases in cell proliferation and matrix production in bones from males and females, as indicated by incorporation of [3H]thymidine into DNA and [3H]proline into collagen. E2 and DHT both increased [3H]thymidine and [3H]proline incorporations, E2 producing greater increases in females than in males. Indomethacin abrogated the effects of loading, but had no effects on those of sex hormones. Loading, or prostanoids, together with sex hormones, produced responses generally equal to or greater than the addition of the individual influences acting independently. In females there was a synergistic response in [3H]thymidine incorporation between loading and E2, which was quantitatively similar to the interaction between E2 and PGE2 or PGI2. The interaction between loading and E2 for [3H]proline incorporation was not mimicked by these prostanoids. In males the synergism in [3H]proline incorporation seen between loading and DHT was mimicked by that between PGI2 and DHT. We conclude that loading stimulates increased bone cell proliferation and matrix production in situ through a prostanoid-dependent mechanism. This response is equal in size in males and females. Estrogen and testosterone increase proliferation and matrix production through a mechanism independent of prostanoid production. The interactions between loading and hormones are reproduced in some but not all cases by E2 and prostaglandins. E2 with loading and prostaglandins has greater effects in female bones, while DHT with loading and prostaglandins has greater effects in males.

Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture.

Mechanical loading of bone explants stimulates prostaglandin E2 (PGE2) and prostacyclin (PGI2) release and increases glucose 6-phosphate dehydrogenase (G6PD) activity. This response is blocked by indomethacin and imitated by exogenous PGs. In the experiments reported here, primary cultures of rat long bone-derived osteoblast-like cells were exposed to a dynamic strain and exogenous PGs in the culture dish. Strain (3400 mu epsilon, 600 cycles, 1 Hz) caused an immediate release of PGI2 into the culture medium but had no effect on PGE2. Strain also caused an increase in G6PD activity per cell and an increase in the smallest transcript of insulin-like growth factor II (IGF-II) (IGF-II T3) but had no effect on the expression of transforming growth factor-beta1 (TGF-beta1). Indomethacin inhibited strain-induced release of PGI2 and suppressed strain-induced stimulation of IGF-II T3 transcript. PGI2 (1 microM) increased G6PD activity and mRNA levels of all three transcripts of IGF-II but had no effect on the mRNA levels of IGF-I or TGF-beta1. PGE2 (1 microM) stimulated G6PD activity and caused a marked increase in IGF-I and the largest transcript of IGF-II (IGF-II T1) but had no effect on the IGF-II transcripts T2 and T3 or on TGF-beta1 mRNA levels. These findings show similarities in response between osteoblast-like cells strained in monolayer culture and bone cells in loaded bone explants in situ. They provide support for a role for IGF-II and PGI2 in the early strain-related response of osteoblasts in loading-related bone modeling/remodeling.

Mechanical loading and sex hormone interactions in organ cultures of rat ulna.

The separate and combined effects of loading and 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) on [3H]thymidine and [3H]proline incorporation were investigated in cultured ulna shafts from male and female rats. Ulnae were cultured and loaded to produce physiological strains in the presence or absence of 10(-8) M E2 or DHT. Loading engendered similar increases in incorporation of [3H]thymidine and [3H]proline in male and female bones. E2 engendered greater increases in incorporation in females than in males, and DHT greater increases in males than in females. In males E2 with loading produced increases in both [3H]thymidine and [3H]proline incorporation, which approximated to the arithmetic addition of the increases due to E2 and loading separately. In females E2 with loading produced increases greater than those in males, and substantially greater than the addition of the effects of E2 and loading separately. Loading with DHT in males also showed additional [3H]thymidine and [3H]proline incorporation. In females there was additional incorporation of [3H]proline, but not [3H]thymidine. The location of incorporation of [3H]thymidine and [3H] proline was consistent with their level of incorporation reflecting periosteal osteogenesis, in which case the early osteogenic effects of sex hormones are gender-specific when acting alone and in combination with loading. In males the effects of estrogen and testosterone add to, but do not enhance, the osteogenic responses to loading. In females testosterone with loading produces an additional effect on [3H]proline incorporation but no greater effect than loading alone on that of [3H]thymidine. In contrast, estrogen and loading together produce a greater effect than the sum of the two influences separately. Because premenopausal bone mass will have been achieved under the influence of loading and estrogen acting together, these findings suggest that the bone loss which follows estrogen withdrawal may result, at least in part, from reduction in the effectiveness of the loading-related stimulus on bone cell activity. This stimulus is normally responsible for maintaining bone mass and architecture.

[Effect of alpha 1-adrenoceptor subtypes on beta-adrenoceptor mediated positive inotropic response in rat left atria].

The distribution of the alpha 1-adrenoceptor (alpha 1-AR) subtypes and the effects of activation of alpha 1-AR subtypes on the beta-adrenoceptor (beta-AR) mediated positive inotropic response were investigated. The radioligand binding assays indicated that the Bmax and Kd values were 11.7 +/- 18 fmol/mg.protein and 86.0 +/- 9.6 pmol/L respectively. Pretreatment of the preparations with 20 mumol/L chloroethylclonidine (CEC) which inactivated alpha 1B subtype, decreased the Bmax to 45.7 +/- 5.2 fmol/mg.protein (P < 0.01). The inhibition curves of 5-methyl-urapidil were best fitted to two site model and indicated that alpha 1A subtype took 28.5% of total 125IBE specific binding sites. In the functional experiments, norepinephrine (NE) induced a positive inotropic response in a concentration dependent manner by activation of both beta- and alpha 1-AR. The concentration-response curves (CRC) for NE were shifted rightward after the pretreatment of the preparations with 20 mumol/L CEC, but leftward in the presence of 1 nmol/L WB4101. In the presence of 10 mumol/L phentolamine which inactivated both alpha 1-AR subtypes, the CRC for NE were shifted leftward. When alpha 1-AR was activated by phenylephrine the CRC for isoproterenol (selective beta-AR agonist) were shifted rightward. The results suggested that the alpha 1B subtype enhanced while the alpha 1A subtype inhibited the beta-AR mediated positive inotropic response. When both alpha 1A and alpha 1B subtypes were activated simultaneously the alpha 1A subtype showed a dominate role.

[Neuropeptide Y reduces desensitization of alpha 1-adrenoceptors in rat blood vessels].

Vasoconstrictive responses to norepinephrine (NE) in isolated rat aorta and renal artery were respectively taken to represent the biological responses mediated by alpha 1B and alpha 1A subtype adrenoceptor (AR). After blood vessels were incubated with 10 mumol/L NE for 2 h, alpha 1-AR mediated vasocontraction was desensitized significantly, alpha 1B-AR being more marked than alpha 1A-AR. In the presence of 0.5 mumol/L neuropeptide Y (NPY), desensitization of alpha 1B-AR was attenuated significantly. In rat aorta the NE-mediated contraction comprised both phasic and tonic elements. After the incubation with NE, the phasic contraction period was prolonged but magnitude decreased, while no change was found for the tonic contraction. In the presence of NPY, the changes of phasic contraction caused by NE preincubation disappeared. It is suggested that retardation and reduction of intracellular Ca2+ release were involved in the mechanism for desensitization of alpha 1B-AR.

Estrogen enhances the stimulation of bone collagen synthesis by loading and exogenous prostacyclin, but not prostaglandin E2, in organ cultures of rat ulnae.

The shafts of ulnae from 110 g male rats were cultured, and after a period of 5 h preincubation one of each pair of bones was either loaded cyclically (500 g, 1 Hz, 8 minutes) to produce physiologic strains (-1300 mu epsilon) or treated with exogenous prostacyclin (PGI2) or prostaglandin E2 (10(-6) M, 8 minutes) in the presence or absence of 17 beta-estradiol (10(-8) M). PGI2, PGE2, and loading stimulated almost immediate increases in glucose 6-phosphate dehydrogenase (G6PD) activity in osteocytes and osteoblasts. This increase was uniform throughout the section with exogenous PGs in the medium but was related to local strain magnitude in loading. Elevated G6PD levels in response to loading and PGI2 persisted for 18 h, by which time, ALP activity in surface osteoblasts was elevated and [3H]proline incorporation into collagen increased. PGE2 produced similar immediate and sustained increases in G6PD activity and [3H]proline incorporation after 18 h but no change in ALP activity. Bones cultured for 18 h with 17 beta-estradiol increased their [3H]proline incorporation, as did those loaded, and treated with PGI2 and PGE2. Loading and PGI2 but not PGE2 produced similar proportional increases in [3H]proline incorporation above the increased baseline of estradiol alone. These results suggest that estrogen and loading together produce a greater osteogenic response than either separately. If so, estrogen withdrawal would result in a rapid fall in bone mass to establish a new equilibrium appropriate to the reduced effectiveness of the loading-related stimulus. Such a fall in bone mass is a characteristic feature of estrogen withdrawal at the menopause.