Human osteoblasts from younger normal and osteoporotic donors show differences in proliferation and TGFβ-release in response to cyclic strain

Mechanical stimulation of bone tissue by physical activity stimulates bone formation in normal bone and may attenuate bone loss of osteoporotic patients. However, altered responsiveness of osteoblasts in osteoporotic bone to mechanical stimuli may contribute to osteoporotic bone involution. The purpose of the present study was to investigate whether osteoblasts from osteoporotic patients and normal donors show differences in proliferation and TGFβ production in responses to cyclic strain. Human osteoblasts isolated from collagenase-treated bone explants of 10 osteoporotic patients (average age 70 ± 6 yr) and 8 normal donors (average age 54 ± 10 yr) were plated into elastic rectangular silicone dishes. Subconfluent cultures were stimulated by cyclic strain (1%, 1 Hz) in an electromechanical cell stretching apparatus at three consecutive days for each 30 min. The cultures were assayed for proliferation, alkaline phosphatase activity and TGFβ release in each three parallel cultures. In all experiments, osteoblasts grown in the same elastic dishes but without mechanical stimulation served as controls. Significant differences between stimulated cultures and unstimulated controls were determined by a paired two-tailed Wilcoxon test. In comparison to the unstimulated controls, osteoblasts from normal donors significantly increased proliferation (p = 0.025) and TGFβ secretion (p = 0.009) into the conditioned culture medium. In contrast, osteoblasts from osteoporotic donors failed to increase both proliferation (p > 0.05) and TGFβ release (p > 0.05) in response to cyclic strain. Alkaline phosphatase activity was not significantly affected (p > 0.05) in normal as well as osteoporotic bone derived osteoblasts.

These findings suggest a different responsiveness to 1% cyclic strain of osteoblasts isolated from normal and osteoporotic bone that could be influenced by both the disease of osteoporosis and the higher average age of the osteoporotic patient group. While osteoblasts from osteoporotic donors failed to increase proliferation and TGFβ release under the chosen mechanical strain regimen that stimulated both parameters in normal osteoblasts, it is possible that some other strain regimen would provide more effective stimulation of osteoporotic cells.     

Differential activation of ERK1,2 MAP kinase signaling pathway in mesenchymal stem cell from control and osteoporotic postmenopausal women

Osteoblasts, the cells responsible for bone formation, derive from mesenchymal stem cells (MSCs) in bone marrow. To acquire a new cell phenotype, uncommitted MSCs must undergo several proliferation and differentiation changes. Although, it is known that extracellular signal-regulated protein kinases (ERKs) mitogen-activated protein (MAP) kinase pathway signaling is involved in the proliferation and differentiation processes, the role of ERKs in osteogenic differentiation it is controversial, at present. In addition, the function that ERK could play in MSCs derived from osteoporotic patients it is not well documented. In this study, we analyze whether previously observed differences in the dynamic response of MSCs from normal and osteoporotic postmenopausal women can be explained by changes in the activation of this signal transduction pathway. Levels of ERK phosphorylation and their correlation with osteogenic differentiation were evaluated in cultures of MSCs derived from osteoporotic postmenopausal women and "healthy" controls. The results show that, under basal conditions, MSCs derived from osteoporotic donors show a level of ERK phosphorylation 2.5 times higher than MSCs derived from control donors. The addition of the osteogenic stimulus only slightly increases the p-ERK level in cells derived from osteoporotic donors, and is higher in cells derived from control women. Important differences in the ability of PD98059 to inhibit phosphorylation of ERK in both types of cells were also observed, as well as the effect that this inhibition produced on calcium deposition. We conclude that the MAP kinase pathway signaling is differentially activated in MSCs derived from osteoporotic postmenopausal women. The high p-ERK levels in MSC derived from osteoporotic donors could determine the unresponsiveness of these cells to the osteogenic differentiation stimulus.     

Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expression in vitro

In a previous study of the cellular mechanism underlaying Wolff's law we showed that mechanical stimulation by intermittent hydrostatic compression (IHC) increases bone formation in cultured fetal mouse calvariae compared to non-stimulated cultures. To test whether mechanical stimuli may modulate bone-specific gene expression, we studied the effect of IHC on alkaline phosphatase (AP) expression and enzyme activity as well as collagen and actin mRNA levels in neonatal mouse calvariae and calvarial bone cells. Two cell populations, one resembling osteoprogenitor (OPR) cells and another resembling osteoblasts (OB) were obtained from calvariae by sequential digestion. IHC was applied by intermittently (0.3 Hz) compressing the gas- phase of a closed culture chamber (peak stress 13 kPa, peak stress rate 32.5 kPas⁻¹).

In control cultures of calvariae as well as OB and OPR cells, AP activity and AP-, collagen-, and actin-mRNA levels all decreased after one or more days, with the exception of OPR cell collagen expression which increased during culture. IHC treatment upregulated AP, collagen and actin expression and AP activity in calvariae and OB cells, but decreased collagen expression in OPR cells.

These results suggest that treatment with IHC promotes the osteoblastic phenotype in bone organ cultures and in osteoblasts. Osteoprogenitor cells seem to react somewhat differently to mechanical stress than osteoblasts. The loss of bone-specific gene expression under control culture conditions, in the absence of mechanical stimuli, suggests that the mechanical environment is important in maintaining the differentiated phenotype of bone cells, and that IHC treatment partially restores this environment in bone cell- and organ cultures.     

Interferon-associated, dsRNA-dependent enzyme activities in a mutant 3T6 cell engaged in the semiconstitutive synthesis of interferon

Cytoplasmic extracts of untreated cultures of a virus-resistant mutant of mouse 3T6 cells, designated 3T6-VrB2, contain two double-stranded, RNA-activated enzyme activities associated with interferon action. These are the synthesis of a low molecular weight oligonucleotide inhibitor of cell-free protein synthesis from ATP, and the phosphorylation of a 67,000 dalton polypeptide by transfer of the gamma phosphate of ATP. Basal levels of both enzyme activities are detectable in extracts of untreated parental 3T6 cells, and are greatly enhanced upon interferon pretreatment. A procedure was developed, using a nonionic detergent to effect cell lysis, which allowed the analysis of the protein kinase activity from as few as 2 x 10(7) cells. Using this procedure, direct proportionalities were demonstrated between the concentration of interferon to which 3T6 cells were exposed, and both the level of protein kinase activity and the magnitude of the antiviral state were established in these cells. Furthermore, untreated cultures of 3T6-VrB2 exhibited both an antiviral state and an intracellular protein kinase activity equal to that of cultures of the parental 3T6 cells pretreated with a single concentration of mouse interferon.     

Effect of bone morphogenetic proteins-4, -5 and -6 on DNA synthesis and expression of bone-related proteins in cultured human periodontal ligament cells

Bone morphogenetic proteins (BMPs) have multiple functions in the development and growth of skeletal and extraskeletal tissues. Therefore, BMPs may regulate the regeneration of periodontal tissue. To investigate this issue, we examined the effects of BMP-4, -5 and -6 on DNA synthesis and the expression of bone-related proteins in cultures of human periodontal ligament (HPL) cells. The expression of bone-related proteins was determined by Real-time polymerase chain reaction and enzyme linked immunosorbent assay in cultures of HPL cells. DNA synthesis was estimated by measuring bromoderoxyuridine incorporation. It was found that BMP-4, -5 and -6 enhanced DNA synthesis dose-dependently. BMP-4 and -5 increased the levels of osteopontin, BMP-2, alkaline phosphatase and core binding factor alpha 1 mRNAs. BMP-6 stimulated the expression of osteopontin, BMP-2, ALPase and osteoprotegerin. These findings show that BMP-4, -5 and -6 have different actions on the expression of bone-related proteins and may play a role in the regeneration of periodontal tissue by promoting cell proliferation and protein expression.     

Differences in zinc status between patients with osteoarthritis and osteoporosis

Zinc has been suggested to play an important role in the development of osteoporosis, whereas the influence of zinc on osteoarthritis has attracted much less attention. The aim of the study was to investigate and compare the zinc status and bone turnover, density, and biomechanical properties of osteoarthritic and osteoporotic patients. The study comprised 40 women who underwent hip replacement due to osteoarthritis or osteoporosis. Serum and urine zinc content, and bone resorption markers and serum bone formation markers were determined. The unaffected hip and the exarticulated affected femoral head underwent DEXA scanning. Bone biopsies were obtained from the femoral heads and the biomechanical properties were determined. The biopsies were ashed and the bone zinc content was ascertained. Osteoarthritic patients had significantly higher serum zinc concentrations and lower urine zinc concentrations than osteoporotic patients, whereas the bone zinc content did not differ. The zinc status was not found to be a predictor for the bone strength. In conclusion, the finding that the zinc status of osteoporotic patients is significantly different from that of osteoartritic patients is new and supports the view that osteoporosis and osteoarthritis rarely occur in the same individual.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

Induction of DNA polymerase α in senescent cultures of normal and Werner's syndrome cultured skin fibroblasts

DNA polymerase α activity was determined following serum stimulation of early and late passages of human diploid fibroblast-like (HDFL) cultures derived from apparently normal donors (two strains) and from a patient with Werner's syndrome (one strain). Induction of this enzyme was observed in both low passage, actively proliferating cultures and in postmitotic “senescent” cultures from all three strains. The maximal polymerase activity of early and late passage cells of each strain were nearly identical when normalized to the number of cells present. However, the activity of the enzyme was observed to be significantly lower in late passage cultures when normalized to total protein content apparently because of enlargement of the senescent cells. The behavior of Werner derived cells was similar to that of the normal cells. The induction of DNA polymerase α in senescent cultures indicates that they retain the capacity to carry out some complex metabolic responses to mitogen stimulation. In addition, these results suggest the possibility that dilution of DNA polymerase α and/or other DNA replication factors may play a role in the onset or maintenance of the postmitotic state in the enlarged senescent HDFL cells.     

Mutation of Prkar1a causes osteoblast neoplasia driven by dysregulation of protein kinase A

Carney complex (CNC) is an autosomal dominant neoplasia syndrome caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A (PKA). This genetic defect induces skin pigmentation, endocrine tumors, myxomas, and schwannomas. Some patients with the complex also develop myxoid bone tumors termed osteochondromyxomas. To study the link between the PRKAR1A mutations and tumor formation, we generated a mouse model of this condition. Prkar1a(+/-) mice develop bone tumors with high frequency, although these lesions have not yet been characterized, either from human patients or from mice. Bone tumors from Prkar1a(+/-) mice were heterogeneous, including elements of myxomatous, cartilaginous, and bony differentiation that effaced the normal bone architecture. Immunohistochemical analysis identified an osteoblastic origin for the abnormal cells associated with islands of bone. To better understand these cells at the biochemical level, we isolated primary cultures of tumoral bone and compared them with cultures of bone from wild-type animals. The tumor cells exhibited the expected decrease in Prkar1a protein and exhibited increased PKA activity. At the phenotypic level, we observed that tumor cells behaved as incompletely differentiated osteoblasts and were able to form tumors in immunocompromised mice. Examination of gene expression revealed down-regulation of markers of bone differentiation and increased expression of locally acting growth factors, including members of the Wnt signaling pathway. Tumor cells exhibited enhanced growth in response to PKA-stimulating agents, suggesting that tumorigenesis in osteoblast precursor cells is driven by effects directly mediated by the dysregulation of PKA.     

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines.

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro proliferation and differentiation potential of bone marrow-derived mesenchymal stem cells from ovariectomized rats

Bone marrow-derived mesenchymal stem cells (BMMSCs) from the patients suffering from age-related osteoporosis were found to have numerous degeneration, such as decreased growth rate, impaired capacity of differentiating into local tissue, and repressed telomerase activity. However, it is not clear whether post-menopausal osteoporotic bone is either subject to such decline in cellular function. In the present study, bone marrow cells were harvested from ovariectomized (OVX) and Sham rats and cultured in vitro at 3 months post-surgery. MTT assay indicated that the proliferation potential of ^OVXBMMSCs was always higher than that of ^ShamBMMSCs, no matter cultured in basic, osteoblastic or adipogenic medium. Alkaline phosphatase activity assay, Alizarin red S staining, Oil red O staining and real-time RT-PCR analysis further demonstrated that bilateral ovariectomization positively influenced the osteoblastic and adipogenic differentiation potential of BMMSCs, this action may be partly mediated through up-regulation of osteoblastic special markers core binding factor a1, collagen type I and low-density lipoprotein receptor-related protein 5, as well as adipogenic special markers peroxisome proliferators activated receptor gamma, CCAAT/enhancer binding protein alpha and adipocyte lipid-binding protein 2. These results may hold great promise for using post-menopausal osteoporotic bone as an attractive autologous marrow source for tissue engineering and cell-based therapies.     

Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow

Although it is well accepted that bone tissue metabolism is regulated by external mechanical loads, it remains unclear to what load-induced physical signals bone cells respond. In this study, a novel computer-controlled stretch device and parallel plate flow chamber were employed to investigate cytosolic calcium (Ca2+i) mobilization in response to a range of dynamic substrate strain levels (0.1-10 percent, 1 Hz) and oscillating fluid flow (2 N/m2, 1 Hz). In addition, we quantified the effect of dynamic substrate strain and oscillating fluid flow on the expression of mRNA for the bone matrix protein osteopontin (OPN). Our data demonstrate that continuum strain levels observed for routine physical activities (< 0.5 percent) do not induce Ca2+i responses in osteoblastic cells in vitro. However, there was a significant increase in the number of responding cells at larger strain levels. Moreover, we found no change in osteopontin mRNA level in response to 0.5 percent strain at 1 Hz. In contrast, oscillating fluid flow predicted to occur in the lacunar-canalicular system due to routine physical activities (2 N/m2, 1 Hz) caused significant increases in both Ca2+i and OPN mRNA. These data suggest that, relative to fluid flow, substrate deformation may play less of a role in bone cell mechanotransduction associated with bone adaptation to routine loads.     

Effect of nitrogen source on the levels of nitrate reductase in the yeast Hansenula anomala.

Levels of nitrate reductase (NR) protein in Hansenula anomala and Hansenula wingei were determined using specific antiserum raised against the enzyme from H. anomala. Extracts from nitrate-grown cells contained NR protein, while in those from cells grown on ammonium, glutamine or peptone, no cross-reacting material could be observed. Enzyme activity correlated with the levels of cross-reacting material. When nitrate was used as nitrogen source, NR was always present, even in cultures with ammonium, glutamine or peptone, although in these cases both the levels of activity and protein were lower. NR activity was consistently two to four times higher in cells grown in glucose than in cells grown in ethanol. Nitrate was required for NR induction, and deprivation of nitrate from nitrate-grown cells resulted in a rapid loss of NR activity.     

Cyclic strain stimulates proliferative capacity, alpha2 and alpha5 integrin, gene marker expression by human articular chondrocytes propagated on flexible silicone membranes

Chondrocytes comprise less than 10% of cartilage tissue but are responsible for sensing and responding to mechanical stimuli imposed on the joint. However, the effect of mechanical signals at the cellular level is not yet fully defined. The purpose of this study was to test the hypothesis that mechanical stimulation in the form of cyclic strain modulates proliferative capacity and integrin expression of chondrocytes from osteoarthritic knee joints. Chondrocytes isolated from articular cartilage during total knee arthroplasty were propagated on flexible silicone membranes. The cells were subjected to cyclic strain for 24 h using a computer-controlled vacuum device, with replicate samples maintained under static conditions. Our results demonstrated increase in proliferative capacity of the cells subjected to cyclic strain compared with cells maintained under static conditions. The flexed cells also exhibited upregulation of the chondrocytic gene markers type II collagen and aggrecan. In addition, cyclic strain resulted in increased expression of the alpha2 and alpha5 integrin subunits, as well as an increased expression of vimentin. There was also intracellular reconfiguration of the enzyme protein kinase C. Our findings suggest that these molecules may play a role in the signal transduction pathway, eliciting cellular response to mechanical stimulation.     

Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains

Summary In submerged cultures of Claviceps sp. CP II, elymoclavine was synthesized only by the growing mycelium (phase P1), whereas cultures of C. purpurea strain 129 produced agroclavine after vegetative growth had also ceased (phase P2). In strain CP II, the peak of activity of malate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphatases was related to the time of maximum growth rate and alkaloid production. Citrate synthase activity paralleled the course of alkaloid synthesis. Strain 129 exhibited a further activity peak of the same magnitude during phase P2. ATP levels in both cultures corresponded to the pattern of change in enzyme activities. Strain CP II contained roughly twice as much orthophosphate and ATP in its cells as strain 129 and exhibited higher average activity of glucose-6-phosphate dehydrogenase. It follows from these results that alkaloid synthesis requires the processes of primary metabolism, even when it occurs after active growth of the culture has ceased. Cultures producing alkaloids oxidized at C-8 exhibit higher glucose-6-phosphate dehydrogenase activity, probably because of a higher NADPH consumption.     

Stimulation of osteoblast growth by an electromagnetic field in a model of bone-like construct

The histogenesis of bone tissue is strongly influenced by physical forces, including magnetic fields. Recent advances in tissue engineering has permitted the generation of three dimensional bone-like constructs. We have investigated the effects of electromagnetic stimulation on human osteoblast cells grown in a hydrophobic polyurethane scaffold. Bone-like constructs were stimulated by pulsed electromagnetic fields in a bioreactor. Proliferation, bone protein expression and calcified matrix production by osteoblasts were measured using histochemical methods. In stimulated cultures, the number of cells was significantly higher compared to static (control) cultures. In both stimulated and control cultures, cells were immunoreactive to osteoblast markers, including type-I collagen, osteocalcin and osteopontin, thus suggesting that the expression of bone-related markers was maintained throughout the in vitro experiments. Morphometric analysis of von Kossa-stained sections revealed that stimulation with electromagnetic field significantly increased matrix calcification. The data lend support to the view that the application of a magnetic field can be used to stimulate cell growth in bone-like constructs in vitro. This finding may be of interest for the production of biomaterials designed for clinical applications.     

Effect of mechanical stimulation on the production of soluble bone factors in cultured fetal mouse calvariae

Mechanical stimulation by intermittent compressive force (ICF) stimulates bone formation and inhibits bone resorption in cultured fetal mouse bone. Fetal bone tissue can produce autocrine factors that stimulate bone cell replication and matrix formation, and paracrine factors that increase the formation of osteoclast precursor-like cells from bone marrow. In the present study, we have tested whether ICF affects the production of such local factors in fetal mouse calvariae. Calvariae were cultured for 4 days in the presence and absence of ICF (130 mbar, 0.3 Hz). Conditioned medium was collected daily and pooled. We found that conditioned medium from ICF-exposed cultures stimulated [³H]-TdR incorporation into DNA, and [³H]-proline incorporation into collagenase digestible protein but not into non-collagen protein in fresh calvarial cultures. Treatment with conditioned medium from ICF-exposed cultures had earlier effects on [³H]-TdR and [³H]-proline incorporation than direct treatment with ICF. Conditioned medium from ICF-exposed cultures decreased the number of osteoclast precursor-like cells in bone marrow cultures stained for tartrate-resistant acid phosphatase. We conclude that ICF stimulates the release (activity) of an autocrine growth-factor from bone. In addition, ICF can stimulate the release (activity) of a paracrine factor, inhibiting the growth and/or differentiation of osteoclast precursor-like cells. These data suggest that mechanical forces may modulate skeletal (re)modeling by affecting the production of local growth factors.     

Molecular basis of alpha-methyltryptophan resistance in amt-1, a mutant of Arabidopsis thaliana with altered tryptophan metabolism.

A mutant of Arabidopsis thaliana, amt-1, was previously selected for resistance to growth inhibition by the tryptophan analog alpha-methyltryptophan. This mutant had elevated tryptophan levels and exhibited higher anthranilate synthase (AS) activity that showed increased resistance to feedback inhibition by tryptophan. In this study, extracts of the mutant callus exhibited higher AS activity than wild-type callus when assayed with either glutamine or ammonium sulfate as amino donor, thus suggesting that elevated AS activity in the mutant was due to an alteration in the alpha subunit of the enzyme. The mutant also showed cross-resistance to 5-methylanthranilate and 6-methylanthranilate and mapped to chromosome V at or close to ASA1 (a gene encoding the AS alpha subunit). ASA1 mRNA and protein levels were similar in mutant and wild-type leaf extracts. Levels of ASA1 mRNA and protein were also similar in callus cultures of mutant and wild type, although the levels in callus were higher than in leaf tissue. Sequencing of the ASA1 gene from amt-1 revealed a G to A transition relative to the wild-type gene that would result in the substitution of an asparagine residue in place of aspartic acid at position 341 in the predicted amino acid sequence of the ASA1 protein. The mutant allele in strain amt-1 has been renamed trp5-1.     

Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces

Summary We report the characterization of human osteoblastic cells that were derived from the surface of trabecular bone fragments. After removal of bone marrow cells, the bone lining osteoblastic cells lining the bone surface were obtained by migration and proliferation from the trabecular surface onto a nylon mesh. The isolated population proliferated in culture and exhibited osteoblastic phenotype. Cultured cells show a regular arrangment in vitro and exhibited multiple interconnecting junctions on scanning electron microscopic examination. Immunocytochemical staining showed that the cells produced almost exclusively type I collagen. Bone-surface-derived cells responded to 1–34 human parathyroid hormone by increasing intracellular cyclic AMP. Cell cultures exhibited high alkaline phosphatase activity, which was unaffected by 1,25 (OH)₂ vitamin D. Untreated cells produced high levels of osteocalcin, a bone-specific protein, and they responded to 1,25(OH) vitamin D by increasing osteocalcin synthesis in a dose-dependent manner. Although cells cultured for up to 5 mo. still produced osteocalcin, the response to 1,25(OH)₂D decreased after multiple passages. This study shows that the bone cell populations isolated from trabecular bone surface are enriched in osteoblast precursors and mature osteoblstic cells.     

Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha

The Wnt/beta-catenin pathway has been implicated in bone cell response to their mechanical environment. This response is the origin of the mechanism by which bone cells adjust bone architecture to maintain bone strength. Osteoporosis is the most widespread failure of this mechanism. The degree of osteoporotic bone loss in men and women is related to bio-available estrogen. Here we report that in osteoblastic ROS 17/2.8 cells and primary osteoblast cultures, a single short period of dynamic mechanical strain, as well as the glycogen synthase kinase-3beta (GSK-3beta) inhibitor LiCl, increased nuclear accumulation of activated beta-catenin and stimulated TCF/LEF reporter activity. This effect was blocked by the estrogen receptor (ER) modulators ICI 182,780 and tamoxifen and was absent in primary osteoblast cultures from mice lacking ERalpha. Microarray expression data for 25,000 genes from total RNA extracted from tibiae of wild-type mice within 24 h of being loaded in vivo showed differential gene regulation between loaded and contralateral non-loaded bones of 10 genes established to be involved in the Wnt pathway. Only 2 genes were involved in loaded tibiae from mice lacking ERalpha (ERalpha(-/-)). Together these data suggest that Wnt/beta-catenin signaling contributes to bone cell early responses to mechanical strain and that its effectiveness requires ERalpha. Reduced effectiveness of bone cell responses to bone loading, associated with estrogen-related decline in ERalpha, may contribute to the failure to maintain structurally appropriate bone mass in osteoporosis in both men and women.