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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts

The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines. Copyright 1999 Academic Press.

Non-NASA Center↗
NASA NTRS1999

Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast. Copyright 1999 Wiley-Liss, Inc.

Non-NASA Center↗
NASA NTRS1999

Calcium metabolism before, during, and after a 3-mo spaceflight: kinetic and biochemical changes

The loss of bone during spaceflight is considered a physiological obstacle for the exploration of other planets. This report of calcium metabolism before, during, and after long-duration spaceflight extends results from Skylab missions in the 1970s. Biochemical and endocrine indexes of calcium and bone metabolism were measured together with calcium absorption, excretion, and bone turnover using stable isotopes. Studies were conducted before, during, and after flight in three male subjects. Subjects varied in physical activity, yet all lost weight during flight. During flight, calcium intake and absorption decreased up to 50%, urinary calcium excretion increased up to 50%, and bone resorption (determined by kinetics or bone markers) increased by over 50%. Osteocalcin and bone-specific alkaline phosphatase, markers of bone formation, increased after flight. Subjects lost approximately 250 mg bone calcium per day during flight and regained bone calcium at a slower rate of approximately 100 mg/day for up to 3 mo after landing. Further studies are required to determine the time course of changes in calcium homeostasis during flight to develop and assess countermeasures against flight-induced bone loss.

NASA Discipline Regulatory Physiology↗
NASA NTRS1999

Skeletal unloading causes resistance of osteoprogenitor cells to parathyroid hormone and to insulin-like growth factor-I

Skeletal unloading decreases bone formation and osteoblast number in vivo and decreases the number and proliferation of bone marrow osteoprogenitor (BMOp) cells in vitro. We tested the ability of parathyroid hormone (PTH) to stimulate BMOp cells in vivo by treating Sprague Dawley rats (n = 32) with intermittent PTH(1-34) (1 h/day at 8 microg/100 g of body weight), or with vehicle via osmotic minipumps during 7 days of normal weight bearing or hind limb unloading. Marrow cells were flushed from the femur and cultured at the same initial density for up to 21 days. PTH treatment of normally loaded rats caused a 2.5-fold increase in the number of BMOp cells, with similar increases in alkaline phosphatase (ALP) activity and mineralization, compared with cultures from vehicle-treated rats. PTH treatment of hind limb unloaded rats failed to stimulate BMOp cell number, ALP activity, or mineralization. Hind limb unloading had no significant effect on PTH receptor mRNA or protein levels in the tibia. Direct in vitro PTH challenge of BMOp cells isolated from normally loaded bone failed to stimulate their proliferation and inhibited their differentiation, suggesting that the in vivo anabolic effect of intermittent PTH on BMOp cells was mediated indirectly by a PTH-induced factor. We hypothesize that this factor is insulin-like growth factor-I (IGF-I), which stimulated the in vitro proliferation and differentiation of BMOp cells isolated from normally loaded bone, but not from unloaded bone. These results suggest that IGF-I mediates the ability of PTH to stimulate BMOp cell proliferation in normally loaded bone, and that BMOp cells in unloaded bone are resistant to the anabolic effect of intermittent PTH therapy due to their resistance to IGF-I.

NASA Discipline Musculoskeletal↗
NASA NTRS1998

The effects of twelve weeks of bed rest on bone histology, biochemical markers of bone turnover, and calcium homeostasis in eleven normal subjects

This study was undertaken to examine the effects of 12 weeks of skeletal unloading on parameters of calcium homeostasis, calcitropic hormones, bone histology, and biochemical markers of bone turnover in 11 normal subjects (9 men, 2 women; 34 +/- 11 years of age). Following an ambulatory control evaluation, all subjects underwent 12 weeks of bed rest. An additional metabolic evaluation was performed after 12 days of reambulation. Bone mineral density declined at the spine (-2.9%, p = 0.092) and at the hip (-3.8%, p = 0.002 for the trochanter). Bed rest prompted a rapid, sustained, significant increase in urinary calcium and phosphorus as well as a significant increase in serum calcium. Urinary calcium increased from a pre-bed rest value of 5.3 mmol/day to values as high as 73 mmol/day during bed rest. Immunoreactive parathyroid hormone and serum 1,25-dihydroxyvitamin D declined significantly during bed rest, although the mean values remained within normal limits. Significant changes in bone histology included a suppression of osteoblastic surface for cancellous bone (3.1 +/- 1.3% to 1.9 +/- 1.5%, p = 0.0142) and increased bone resorption for both cancellous and cortical bone. Cortical eroded surface increased from 3.5 +/- 1.1% to 7.3 +/- 4.0% (p = 0.018) as did active osteoclastic surface (0.2 +/- 0.3% to 0.7 +/- 0.7%, p = 0.021). Cancellous eroded surface increased from 2.1 +/- 1.1% to 4.7 +/- 2.2% (p = 0.002), while mean active osteoclastic surface doubled (0.2 +/- 0.2% to 0.4 +/- 0.3%, p = 0.020). Serum biochemical markers of bone formation (osteocalcin, bone-specific alkaline phosphatase, and type I procollagen extension peptide) did not change significantly during bed rest. Urinary biochemical markers of bone resorption (hydroxyproline, deoxypyridinoline, and N-telopeptide of type I collagen) as well as a serum marker of bone resorption (type I collagen carboxytelopeptide) all demonstrated significant increases during bed rest which declined toward normal during reambulation. Thus, under the conditions of this study, the human skeleton appears to respond to unloading by a rapid and sustained increase in bone resorption and a more subtle decrease in bone formation.

NASA Discipline Musculoskeletal↗
NASA NTRS1998

Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers

Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study demonstrates that PLGA foams are suitable substrates for osteoblast growth and differentiated function independent of cell source.

Non-NASA Center↗
NASA NTRS1999

Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor

PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

Prostate/chemistry/cytology↗
NASA NTRS1998

Osteoblastic phenotype of rat marrow stromal cells cultured in the presence of dexamethasone, beta-glycerolphosphate, and L-ascorbic acid

We investigated the effects of the time course of addition of osteogenic supplements dexamethasone, beta-glycerolphosphate, and L-ascorbic acid to rat marrow stromal cells, and the exposure time on the proliferation and differentiation of the cells. It was the goal of these experiments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. To determine this, two studies were performed; one study was based on the age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proliferate rapidly at early time points in the presence and absence of osteogenic supplements as determined by 3H-thymidine incorporation into the DNA of replicating cells. These results were supported by cell counts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented medium immediately upon harvest. The ALP levels at 21 days were six times greater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reaching an absolute value of 75 x 10(-7) micromole/min/cell. Osteocalcin production reached 20 x 10(-6) ng/cell at 21 days in both studies for cells maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These results suggest that the addition of osteogenic supplements to marrow-derived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat model.

Non-NASA Center↗
NASA NTRS1997

Skeletal unloading inhibits the in vitro proliferation and differentiation of rat osteoprogenitor cells

Loss of weight bearing in the growing rat decreases bone formation, osteoblast numbers, and bone maturation in unloaded bones. These responses suggest an impairment of osteoblast proliferation and differentiation. To test this assumption, we assessed the effects of skeletal unloading using an in vitro model of osteoprogenitor cell differentiation. Rats were hindlimb elevated for 0 (control), 2, or 5 days, after which their tibial bone marrow stromal cells (BMSCs) were harvested and cultured. Five days of hindlimb elevation led to significant decreases in proliferation, alkaline phosphatase (AP) enzyme activity, and mineralization of BMSC cultures. Differentiation of BMSCs was analyzed by quantitative competitive polymerase chain reaction of cDNA after 10, 15, 20, and 28 days of culture. cDNA pools were analyzed for the expression of c-fos (an index of proliferation), AP (an index of early osteoblast differentiation), and osteocalcin (a marker of late differentiation). BMSCs from 5-day unloaded rats expressed 50% less c-fos, 61% more AP, and 35% less osteocalcin mRNA compared with controls. These data demonstrate that cultured osteoprogenitor cells retain a memory of their in vivo loading history and indicate that skeletal unloading inhibits proliferation and differentiation of osteoprogenitor cells in vitro.

NASA Discipline Musculoskeletal↗
NASA NTRS1997

Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

NASA Center ARC↗
NASA NTRS1997

Mineral induction by immobilized phosphoproteins

Dentin phosphoproteins are thought to have a primary role in the deposition of mineral on the collagen of dentin. In this study we determined the type of binding between collagen and phosphoproteins necessary for mineral formation onto collagen fibrils and whether the phosphate esters are required. Bovine dentin phosphophoryn or phosvitin from egg yolk were immobilized on reconstituted skin type I collagen fibrils by adsorption or by covalent cross-linking. In some samples the ester phosphate was removed from the covalently cross-linked phosphoproteins by treatment with acid phosphatase. All samples were incubated at 37 degrees C in metastable solutions that do not spontaneously precipitate. Reconstituted collagen fibrils alone did not induce mineral formation. The phosphoproteins adsorbed to the collagen fibrils desorbed when the mineralization medium was added, and mineral was not induced. The mineral induced by the cross-linked phosphoproteins was apatite, and the crystals were confined to the surface of the collagen fibrils. With decreasing medium saturation the time required for mineral induction increased. The interfacial tensions calculated for apatite formation by either phosphoprotein cross-linked to collagen were about the same as that for phosphatidic acid liposomes and hydroxyapatite. This similarity in values indicates that the nucleation potential of these highly phosphorylated surfaces is about the same. It is concluded that phosphoproteins must be irreversibly bound to collagen fibrils for the mineralization of the collagen network in solutions that do not spontaneously precipitate. The phosphate esters of phosphoproteins are required for mineral induction, and the carboxylate groups are not sufficient.

Non-NASA Center↗
NASA NTRS1997

Synchrony of clonal cell proliferation and contiguity of clonally related cells: production of mosaicism in the ventricular zone of developing mouse neocortex

We have analyzed clonal cell proliferation in the ventricular zone (VZ) of the early developing mouse neocortex with a replication-incompetent retrovirus encoding human placental alkaline phosphatase (AP). The retrovirus was injected into the lateral ventricles on embryonic day 11 (E11), i.e., at the onset of neuronogenesis. Three days postinjection, on E14, a total of 259 AP-labeled clones of various sizes were found in 7 fetal brains. There are approximately 7 cell cycles between E11 and E14 (), and there is a 1-2 cell cycle delay between retroviral injection and the production of a retrovirally labeled "founder" cell; thus, we estimate that the "age" of the clones was about 5-6 cell cycles. Almost one-half of the clones (48.3%) identified were pure proliferating clones containing cells only in the VZ. Another 18.5% contained both proliferating and postproliferative cells, and 33.2% contained only postproliferative cells. It was striking that over 90% of the clonally related proliferating cells occurred in clusters of two or more apparently contiguous cells, and about 73% of the proliferating cells occurred in clusters of three or more cells. Regardless of the number of cells in the clone, these clusters were tightly packed and confined to a single level of the VZ. This clustering of proliferating cells indicates that clonally related cells maintain neighbor-neighbor relationships as they undergo interkinetic nuclear migration and progress through several cell cycles, and, as a result, the ventricular zone is a mosaic of small clusters of clonally related and synchronously cycling cells. In addition, cells in the intermediate zone and the cortical plate were also frequently clustered, indicating that they became postproliferative at a similar time and that the output of the VZ is influenced by its mosaic structure.

NASA Discipline Neuroscience↗
NASA NTRS1996

Fibronectin regulates calvarial osteoblast differentiation

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.

NASA Program Space Physiology and Countermeasures↗
NASA NTRS1995

Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts

A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.

NASA Discipline Musculoskeletal↗
NASA NTRS1995

The molecular response of bone to growth hormone during skeletal unloading: regional differences

Hind limb elevation of the growing rat provides a good model for the skeletal changes that occur during space flight. In this model the bones of the forelimbs (normally loaded) are used as an internal control for the changes that occur in the unloaded bones of the hind limbs. Previous studies have shown that skeletal unloading of the hind limbs results in a transient reduction of bone formation in the tibia and femur, with no change in the humerus. This fall in bone formation is accompanied by a fall in serum osteocalcin (bone Gla protein, BGP) and bone BGP messenger RNA (mRNA) levels, but a rise in bone insulin-like growth factor-I (IGF-I) protein and mRNA levels and resistance to the skeletal growth-promoting actions of IGF-I. To determine whether skeletal unloading also induced resistance to GH, we evaluated the response of the femur and humerus of sham and hypophysectomized rats, control and hind limb elevated, to GH (two doses), measuring mRNA levels of IGF-I, BGP, rat bone alkaline phosphatase (RAP), and alpha 1(1)-procollagen (coll). Hypophysectomy (HPX) decreased the mRNA levels of IGF-I, BGP, and coll in the femur, but was either less effective or had the opposite effect in the humerus. GH at the higher dose (500 micrograms/day) restored these mRNA levels to or above the sham control values in the femur, but generally had little or no effect on the humerus. RAP mRNA levels were increased by HPX, especially in the femur. The lower dose of GH (50 micrograms/day) inhibited this rise in RAP, whereas the higher dose raised the mRNA levels and resulted in the appearance of additional transcripts not seen in controls. As for the other mRNAs, RAP mRNA in the humerus was less affected by HPX or GH than that in the femur. Hind limb elevation led to an increase in IGF-I, coll, and RAP mRNAs and a reduction in BGP mRNA in the femur and either had no effect or potentiated the response of these mRNAs to GH. We conclude that GH stimulates a number of markers of bone formation by raising their mRNA levels, and that skeletal unloading does not block this response, but the response varies substantially from bone to bone.

Non-NASA Center↗
NASA NTRS1994

Signal transduction pathways mediating parathyroid hormone regulation of osteoblastic gene expression

Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. For example, excessive or inappropriate production of PTH or the related hormone, parathyroid hormone related protein (PTHrP), accounts for the majority of the causes of hypercalcemia. Both hormones act through the same receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. Thus, the osteoblast mediates the effect of PTH in the resorption process. In this process, PTH causes a change in the function and phenotype of the osteoblast from a cell involved in bone formation to one directing the process of bone resorption. In response to PTH, the osteoblast decreases collagen, alkaline phosphatase, and osteopontin expression and increases production of osteocalcin, cytokines, and neutral proteases. Many of these changes have been shown to be due to effects on mRNA abundance through either transcriptional or post-transcriptional mechanisms. However, the signal transduction pathway for the hormone to cause these changes is not completely elucidated in any case. Binding of PTH and PTHrP to their common receptor has been shown to result in activation of protein kinases A and C and increases in intracellular calcium. The latter has not been implicated in any changes in mRNA of osteoblastic genes. On the other hand activation of PKA can mimic all the effects of PTH; protein kinase C may be involved in some responses. We will discuss possible mechanisms linking PKA and PKC activation to changes in gene expression, particularly at the nuclear level.

Review↗
NASA NTRS1993

Embryonic mouse pre-metatarsal development in organ culture

Embryonic mouse pre-metatarsals were removed from embryos at 13 days of gestation and cultured in a defined, serum-free medium for up to 15 days. By histological analysis, we observe that the cultured pre-metatarsal tissue undergoes a similar developmental profile as pre-metatarsals growing normally in vivo. The initial mesenchyme condensation regions undergo differentiation and morphogenesis to form distinct rods made up of cartilage tissue. A marker of this differentiation step is the synthesis of type II collagen. Metabolic labelling, pepsin digestion, SDS-PAGE, and autoradiography were used to demonstrate this protein when cartilage tissue is present in the cultures. After additional culture time, terminal chondrocyte differentiation and morphogenesis take place in specific regions of the cartilage rods to form bands of hypertrophied chondrocytes. One marker of this differentiation step is the synthesis of the enzyme alkaline phosphatase. We have measured the activity of this enzyme throughout the culture period and see a substantial increase at the time of terminal chondrocyte differentiation. Another feature of hypertrophied chondrocytes is that the matrix around the cells becomes calcified. Calcified matrix in our cultured pre-metatarsals was visualized by staining with alizarin red. By supplementing the defined culture medium with ITS, we observed that terminal chondrocyte differentiation took place in a shorter culture time. Supplementation of the medium with serum results in a similar acceleration of terminal differentiation, and, with additional culture time, an osteoid-like matrix forms around the central region of the rods.

Non-NASA Center↗
NASA NTRS1992

Studies of matrix vesicle-induced mineralization in a gelatin gel

Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

Non-NASA Center↗