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D D Bikle

Publications and source records attributed to D D Bikle.

At least 73 records · Page 4Linked to original sources
PubMed1994

Skeletal unloading induces resistance to insulin-like growth factor I.

In previous studies with a hindlimb elevation model, we demonstrated that skeletal unloading transiently inhibits bone formation. This effect is limited to the unloaded bones (the normally loaded humerus does not cease growing), suggesting that local factors are of prime importance. IGF-I is one such factor; it is produced in bone and stimulates bone formation. To determine the impact of skeletal unloading on IGF-I production and function, we assessed the mRNA levels of IGF-I and its receptor (IGF-IR) in the proximal tibia and distal femur of growing rats during 2 weeks of hindlimb elevation. The mRNA levels for IGF-I and IGF-IR rose during hindlimb elevation, returning toward control values during recovery. This was accompanied by a 77% increase in IGF-I levels in the bone, peaking at day 10 of unloading. Changes in IGF binding protein levels were not observed. Infusion of IGF-I (200 micrograms/day) during 1 week of hindlimb elevation doubled the increase in bone mass of the control animals but failed to reverse the cessation of bone growth in the hindlimb-elevated animals. We conclude that skeletal unloading induces resistance to IGF-I, which may result secondarily in increased local production of IGF-I.

Aging↗
PubMed1994

Impact of skeletal unloading on bone formation: role of systemic and local factors.

We have developed a model of skeletal unloading using growing rats whose hindlimbs are unweighted by tail suspension. The bones in the hindlimbs undergo a transient cessation of bone growth; when reloaded bone formation is accelerated until bone mass is restored. These changes do not occur in the normally loaded bones of the forelimbs. Associated with the fall in bone formation is a fall in 1,25(OH)2D3 production and osteocalcin levels. In contrast, no changes in parathyroid hormone, calcium, or corticosterone levels are seen. To examine the role of locally produced growth factors, we have measured the mRNA and protein levels of insulin like growth factor-1 (IGF-1) in bone during tail suspension. Surprisingly, both the mRNA and protein levels of IGF-1 increase during tail suspension as bone formation is reduced. Furthermore, the bones in the hindlimbs of the suspended animals develop a resistance to the growth promoting effects of both growth hormone and IGF-1 when given parenterally. Thus, the cessation of bone growth with skeletal unloading is apparently associated with a resistance to rather than failure to produce local growth factors. The cause of this resistance remains under active investigation.

Animals↗
PubMed1994

Production of 1,25 dihydroxyvitamin D3 by perfused pig skin.

Keratinocytes produce large amounts of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) in vitro. 1,25(OH)2D3 is detectable in anephric humans and pigs and can be increased to near-normal levels by vitamin D or 250HD, indicating an extrarenal source. To determine whether the skin is one of these extrarenal sources, we perfused isolated flaps of porcine skin for 8 h with 250HD3 in serum-free medium at 1 ml/min, collecting the venous effluent as 15-min samples. The samples were extracted and the vitamin D metabolites purified by high-performance liquid chromatography and assayed by competitive protein-binding techniques. Production of 1,25(OH)2D3 continued for the duration of the perfusion, tending to increase in the last 2 hours. The amount of 1,25(OH)2D3 produced varied both with time in the same pig skin and between pig skins; maximum production of 1,25(OH)2D3 in these experiments was 8 pg/min. 24,25(OH)2D3 production was higher than 1,25(OH)2D3 production, reaching a maximum rate of 180 pg/min. Considering that the production rate of 1,25(OH)2D3 in humans is 1.25 ng/min and that a 48-cm2 skin flap represents 1/350 the surface area of a human, and assuming that human and pig skin make 1,25(OH)2D3 at comparable rates, one can determine that the skin has the potential to maintain near-normal levels of 1,25(OH)2D3 in the absence of kidneys when provided with adequate substrate.

24,25-Dihydroxyvitamin D 3↗
PubMed1994

Expression of the genes for insulin-like growth factors and their receptors in bone during skeletal growth.

Insulin-like growth factors (IGF) are important regulators of skeletal growth. To determine whether the capacity to produce and respond to these growth factors changes during skeletal development, we measured the protein and mRNA levels for IGF-I, IGF-II, and their receptors (IGF-IR and IGF-IIR, respectively) in the tibia and femur of rats before and up to 28 mo after birth. The mRNA levels remained high during fetal development but fell after birth, reaching a nadir by 3-6 wk. This fall was most pronounced for IGF-II and IGF-IIR mRNA and least pronounced for IGF-I mRNA. However, after 6 wk, both IGF-I and IGF-IR mRNA levels recovered toward the levels observed at birth. In the prenatal bones, the signals for the mRNAs of IGF-II and IGF-IIR were stronger than the signals for the mRNAs of IGF-I and IGF-IR, although the content of IGF-I was three- to fivefold greater than that of IGF-II. IGF-II levels fell postnatally, whereas the IGF-I content rose after birth such that the ratio IGF-I/IGF-II continued to increase with age. We conclude that, during development, rat bone changes its capacity to produce and respond to IGFs with a progressive trend toward the dominance of IGF-I.

Aging↗
PubMed1994

Altered skeletal pattern of gene expression in response to spaceflight and hindlimb elevation.

Spaceflight leads to osteopenia, in part by inhibiting bone formation. Using an animal model (hindlimb elevation) that simulates the weightlessness of spaceflight, we and others showed a reversible inhibition of bone formation and bone mineralization. In this study, we have measured the mRNA levels of insulin-like growth factor I (IGF-I), IGF-I receptor (IGF-IR), alkaline phosphatase, and osteocalcin in the tibiae of rats flown aboard National Aeronautics and Space Administration Shuttle Flight STS-54 and compared the results with those obtained from their ground-based controls and from the bones of hindlimb-elevated animals. Spaceflight and hindlimb elevation transiently increase the mRNA levels for IGF-I, IGF-IR, and alkaline phosphatase but decrease the mRNA levels for osteocalcin. The changes in osteocalcin and alkaline phosphatase mRNA levels are consistent with a shift toward decreased maturation, whereas the rise in IGF-I and IGF-IR mRNA levels may indicate a compensatory response to the fall in bone formation. We conclude that skeletal unloading during spaceflight or hindlimb elevation resets the pattern of gene expression in the osteoblast, giving it a less mature profile.

Alkaline Phosphatase↗
PubMed1994

Role of vitamin D, its metabolites, and analogs in the management of osteoporosis.

Vitamin D and its metabolites are well-established regulators of bone mineral homeostasis. Their clearest role is in the prevention and treatment of rickets and osteomalacia, bone diseases characterized by inadequate bone formation, and mineralization. Much of the effectiveness of vitamin D and its active metabolite 1,25(OH)2D in treating such disorders rests with their ability to increase serum levels of calcium and phosphate principally by stimulating intestinal calcium and phosphate absorption. Osteoporosis is not a disease resulting from obvious deficiencies in vitamin D, calcium, and phosphate. More subtle deficiencies, however, may be found, especially among the elderly with decreased intake of dairy products, reduced sunlight exposure, and less efficient intestinal absorption of bone minerals. Such subtle deficiencies may account for the ability of vitamin D and calcium supplementation to have a beneficial effect on bone mineral density in this population. Estrogen administration to postmenopausal females raises 1,25(OH)2D levels, presumably through increased renal production, and this increase is associated with increased intestinal calcium transport. Serum measurements of the vitamin D metabolites in general, however, and 1,25(OH)2D in particular do not consistently show evidence of a decrease at the time of menopause. Although most studies show a fall in intestinal calcium transport with age, which can be reversed with 1,25(OH)2D or estrogen, even these observations have not been found consistently. Thus, some investigators have addressed the issue of tissue resistance to 1,25(OH)2D and have noted decreased VDR in the intestine and reduced 1,25(OH)2D accumulation by bone with age. Despite no obvious deficiency of vitamin D in most patients with osteoporosis, clinical trials with vitamin D or 1,25(OH)2D show promise. Vitamin D treatment will probably prove most efficacious in populations with marginal vitamin D intake and/or limited sunlight exposure; high doses would not be required, and the treatment would be safe. This would be a physiologic and not a pharmacologic use of vitamin D. The use of 1,25(OH)2D for treatment of osteoporosis in individuals with adequate nutrition and sunlight exposure may require somewhat higher than physiologic doses to be effective. Perhaps such doses are necessary to stimulate osteoblast activity and/or differentiation; by raising the serum calcium level, such doses of 1,25(OH)2D might block its otherwise stimulatory effect on osteoclast number and activity. Such doses run the risk of hypercalcemia and hypercalciuria, leading to nephrolithiasis and/or nephrocalcinosis. These undesirable side effects appear to be less common with the use of 1 alpha OHD compared with 1,25(OH)2D, but this may be because of the lower levels of calcium consumption in Japan where 1 alpha OHD is widely prescribed.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗
PubMed1993

Zipper protein, a newly described tropomyosin-like protein of the intestinal brush border.

We have cloned and sequenced from a chick intestinal library the cDNA for a new tropomyosin-like protein with an extensive leucine zipper motif. The cDNA recognized a 2.5-kilobase transcript with highest levels in the intestine. The open reading frame encoded a protein with 239 residues (28 kDa), the deduced sequence of which forms 27 heptad repeats, 21 of which begin with leucine and the other 6 with conservative substitutions (methionine, valine, threonine). This sequence predicts a coiled coil dimer similar to that of tropomyosin with which it has 34% homology. We have named this newly described protein zipper protein. The protein was expressed in bacteria. Antibodies were made to peptides representing different regions of the deduced sequence and tested for their ability to recognize the recombinant zipper protein on immunoblots. Such antibodies were used to immunolocalize zipper protein to the intestinal brush border. A radioimmunoassay was then established using recombinant zipper protein as standard and tracer and one of the affinity-purified antisera as primary antibody. Extracts from intestine, kidney, and liver displaced tracer zipper protein in parallel with that of the standard curve, and zipper protein levels were readily measured in those tissues to be 2.5 +/- 0.4, 0.34 +/- 0.03, and 0.15 +/- 0.03 micrograms/mg of protein, respectively. Brain contained no detectable zipper protein. We conclude that zipper protein is a tropomyosin-like protein found predominantly in the intestinal brush border; its location and structural similarity to tropomyosin suggest a possible role in regulating the interaction of brush border myosin 1 with the actin core of the microvillus.

Amino Acid Sequence↗
PubMed1993

Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes.

Calcium plays a crucial role in regulating the growth and differentiation of cultured keratinocytes. However, the mechanism(s) of this regulation is not clear. Prior studies have shown that intracellular free calcium (Cai) increases with keratinocyte differentiation. In this study, in order to evaluate the role of cytosolic free calcium and organelle-bound calcium in keratinocyte differentiation, we quantitated and localized calcium pools in keratinocytes, utilizing the fluorescence probe indo-1 and ion-capture cytochemistry, respectively. Cai of undifferentiated keratinocytes was 80-120 nM, whereas Cai of differentiated keratinocytes was 200-300 nM depending on the extent of differentiation. The Cai of individual cells in an undifferentiated colony was heterogeneous (60-160 nM) with larger cells displaying higher Cai. Heterogeneity also was observed in the intracellular calcium-containing precipitates in the different layers of stratifying keratinocyte cultures using the cytochemical technique. Calcium precipitates were abundant in the lower cell layers, progressively decreasing apically, with the uppermost layer devoid of precipitates. Calcium-containing precipitates appeared as fine-to-coarse electron-dense granules on the plasma membrane, within the cytosol, mitochondria, nucleus, and vacuolar organelles. Whereas ionomycin in the presence of extracellular calcium increased the amount of intracellular calcium precipitates, EGTA removed calcium precipitates from organelles. Unlike intact epidermis, keratinocytes displayed no extracellular calcium reservoirs. Putative calcium binding sites, visualized by trivalent lanthanum (La) binding, were abundant on cell membranes and desmosomes of basaloid cells, but decreased in the upper cell layers. These studies revealed differences in the distribution of free ionic calcium (as determined by the fluorescence technique) and organelle-bound calcium (as determined by the cytochemical technique). Striking differences were also observed in calcium localization between intact epidermis and cultured epidermal cells. The localization pattern of calcium in cultured keratinocytes may reflect the hyperproliferative state of these cells, as in psoriatic epidermis, and/or the absence of a normal permeability barrier in these submerged cultures.

Binding Sites↗
PubMed1993

Regulation of keratinocyte growth, differentiation, and vitamin D metabolism by analogs of 1,25-dihydroxyvitamin D.

1,25(OH)2D has numerous actions on many tissues. Analogs of 1,25(OH)2D are being sought that are selective, to further an understanding of the mechanisms of action of 1,25(OH)2D and to improve its therapeutic efficacy. Toward these ends we examined eight analogs of 1,25(OH)2D for their ability to regulate 25OHD metabolism by keratinocytes. Choosing the three most potent, we then examined their ability to inhibit keratinocyte proliferation, stimulate cornified envelope formation (a marker of differentiation), and bind to the 1,25(OH)2D receptor (VDR). 1,25(OH)2-24F2-D, 1,25(OH)2-delta 16-D, and 1,25(OH)2-delta 16,23yne-D proved the most potent in inhibiting 1,25(OH)2D production and stimulating 24,25(OH)2D production, being approximately 10-100 times more potent than 1,25(OH)2D itself. 1,25(OH)2-delta 16-D had the highest affinity for the VDR (fourfold higher than that for 1,25(OH)2D itself) and had the greatest ability both to inhibit proliferation and to stimulate differentiation. 1,25(OH)2-delta 16,23yne-D also had a higher affinity for the VDR but was of less or equal potency in stimulating cornified envelope formation and inhibiting proliferation. 1,25(OH)2-24F2-D, which was the most potent regulator of 25OHD metabolism, had a lower affinity for the VDR and was less potent than 1,25(OH)2D in inhibiting proliferation. Our results indicate that even in the same cell different analogs have different rank orders of potency for the various actions of 1,25(OH)2D.

Calcifediol↗
PubMed1993

Alcohol-induced bone disease: relationship to age and parathyroid hormone levels.

Alcohol abuse leads to osteopenia and fractures. Epidemiological evidence suggests that older alcoholics are at substantially greater risk of fractures than younger alcoholics. To examine the interaction of age and alcohol abuse on bone mineral homeostasis, we studied 27 subjects with a history of 10 more years of alcohol abuse ranging in age from 26-68 years. They were evaluated for disordered bone mineral homeostasis by assessing bone density (by quantitative computed tomography of the lumbar spine), histomorphometry of a transcortical biopsy from the iliac crest, serum levels of vitamin D metabolites and parathyroid hormone, and serum and urine levels of bone minerals. Seventeen of the subjects were found to have spinal compression fractures by routine radiologic procedures. The older the subject the more likely the subject was to have such a fracture. Bone densitometry indicated a marked reduction in spinal bone density with 15 subjects below 2 SD of normal aged-matched controls. Bone density fell sharply with the age of the subject. Histomorphometry of iliac crest bone biopsies revealed no evidence of osteomalacia, but total resorption surfaces were increased. Consistent with the lack of osteomalacia were the normal levels of the vitamin D metabolites. The increased total resorption surfaces were correlated with high normal or elevated levels of parathyroid hormone as indicated both by radioimmunoassay and by urinary cAMP levels. Bone formation and active bone resorption (resorption surfaces containing osteoclasts) did not correlate with parathyroid hormone levels, however, but correlated negatively with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Skeletal unloading decreases production of 1,25-dihydroxyvitamin D.

The plasma concentration of 1,25-dihydroxyvitamin D [1,25(OH)2D] decreases during skeletal unloading and increases when normal weight bearing is restored. To determine whether these changes in plasma 1,25(OH)2D reflect changes in production, metabolic clearance, or both we measured the kinetics of 1,25(OH)2D metabolism in rats whose skeletons were normally loaded, unloaded, or reloaded after a period of nonweight bearing. Skeletal unloading produced a transient but striking fall in the production (-73%) and plasma concentration (-72%) of 1,25(OH)2D without having a significant effect (< 20%) on metabolic clearance. Skeletal reloading returned production to normal. Bone formation predictably decreased during unloading and returned to normal after return to weight bearing. No consistent changes in blood ionized calcium, plasma immunoreactive parathyroid hormone (irPTH), or plasma phosphorus occurred. These data suggest that the changes in plasma 1,25-(OH)2D associated with changes in skeletal weight bearing primarily reflect changes in 1,25(OH)2D production. The data provide no evidence that the changes in 1,25(OH)2D production are a consequence of changes in blood ionized calcium, plasma irPTH, or phosphorus.

Animals↗
PubMed1993

Elevated 1,25-dihydroxyvitamin D levels in patients with chronic obstructive pulmonary disease treated with prednisone.

Glucocorticoid administration is a well established cause of osteopenia. Mechanisms underlying the deleterious effect of glucocorticoids on bone may include direct inhibition of bone formation as well as indirect effects through changes in intestinal calcium absorption, renal calcium excretion, and/or levels of the calciotropic hormones. To further examine the potential role of the calciotropic hormones we measured serum levels of PTH and 1,25 dihydroxyvitamin D [1,25(OH)2D], as well as serum and urine levels of calcium and vertebral bone density in patients with chronic obstructive pulmonary disease being managed with or without prednisone. Patients treated with prednisone had lower spinal bone density (53 vs. 106 mg/cm3) and higher serum calcium (2.40 vs. 2.33 mmol/l), urine calcium (6.9 vs. 2.7 mmol/24h), and 1,25(OH)2D levels (147 vs. 95 pmol/L). Compared to the patients not treated with glucocorticoids. PTH levels also tended to be higher (33 vs. 26 microliters-eq/ml), but the difference was not significant. Serum and urine calcium levels correlated positively with 1,25(OH)2D levels, but none of these measurements correlated with PTH levels. Our results suggest that prednisone treatment alters the regulation of 1,25(OH)2D production, and this may contribute to the loss of bone mineral induced by prednisone.

Aged↗
PubMed1992

Lanthanum influx into cultured human keratinocytes: effect on calcium flux and terminal differentiation.

Trivalent cation lanthanum (La) binds to calcium binding sites of cells and either mimics the properties of calcium or inhibits the effects of calcium by displacing calcium from its binding sites. Extracellular calcium induces differentiation of human epidermal keratinocytes in culture, in part by increasing the intracellular calcium levels (Cai). Therefore, in this study we determined the effect of La on differentiation and intracellular calcium levels of keratinocytes. We observed that La inhibited the production of cornified envelopes, a marker for terminal differentiation of keratinocytes. La inhibited the calcium requiring envelope cross-linking enzyme, transglutaminase, in a direct manner, presumably, by displacing calcium from its binding site on the enzyme. La inhibited the influx and the efflux of 45Ca from keratinocytes. Paradoxically, extracellular La appeared to increase the Cai levels of keratinocytes as measured by the fluorescent probe indo-1. However, subsequent experiments revealed that indo-1 bound La with a higher affinity than Ca and emitted fluorescence in the same wavelength as the Ca bound form. Using this probe, we observed that La enters keratinocytes in a dose-dependent fashion and achieves concentrations exceeding 80 nM when the external La concentration is raised to 300 microM. This fully accounted for the apparent increase in Cai when La was added to the cells. Treatment of cells with ionomycin increased indo-1 fluorescence maximally in the presence of La indicating influx of La via this Ca specific ionophore. Our results indicate that La enters cells and inhibits calcium mediated keratinocyte differentiation both by blocking Ca influx and by blocking calcium regulated intracellular processes such as transglutaminase directed cornified envelope formation.

Biological Transport↗
PubMed1992

Metabolic acidosis reverses the increase in serum 1,25(OH)2D in phosphorus-restricted normal men.

We tested the hypothesis that in humans, metabolic acidosis can disorder the metabolism of 1,25-dihydroxyvitamin D [1,25(OH)2D] by impairing the capacity for a sustained physiological stimulus to increase renal production of this hormone. Specifically, in seven healthy men in whom restriction of dietary phosphorus had doubled their serum concentration of 1,25(OH)2D, we induced metabolic acidosis of moderate severity with oral NH4Cl, administered for 7 days. With induction of acidosis, the serum concentration of 1,25(OH)2D decreased sharply and remained decreased and near constant throughout the period of acidosis, the decrease amounting to one-half of the increment induced by phosphorus restriction alone. The serum concentration of free 1,25(OH)2D also decreased, since the measured free fraction of 1,25(OH)2D was unaffected by NH4Cl. The decrease in serum 1,25(OH)2D was accounted for by a 16% increase in its metabolic clearance rate and by a 19% decrease in its production rate. Metabolic acidosis induced a modest increase in the concentrations of blood ionized calcium and serum phosphorus. Multiple linear regression analysis revealed that serum levels of 1,25(OH)2D varied inversely and significantly with those of plasma hydrogen ion (R = -0.77, P < 0.001), but not with those of blood ionized calcium or serum phosphorus. These data demonstrate in humans that metabolic acidosis can substantially reverse the increase in serum concentration of 1,25(OH)2D induced by phosphorus restriction. The data provide evidence that acidosis can restrict the increase in renal production and serum concentration of 1,25(OH)2D effected by a sustained physiological stimulus.

Acid-Base Equilibrium↗
PubMed1992

Adenosine triphosphate stimulates phosphoinositide metabolism, mobilizes intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.

During wound healing, release of ATP from platelets potentially exposes the epidermis to concentrations of ATP known to alter cellular functions mediated via changes in inositol trisphosphate (IP3) and intracellular calcium (Cai) levels. Therefore, we determined whether keratinocytes respond to ATP with a rise in Cai and IP3 and whether such increases are accompanied by a change in their proliferation and differentiation. Changes in Cai were measured in Indo-1-loaded neonatal human foreskin keratinocytes after stimulation with extracellular ATP. Extracellular ATP evoked a transient and acute increase in Cai of keratinocytes both in the presence and in the absence of extracellular calcium. ATP also induced the phosphoinositide turnover of keratinocytes, consistent with its effect in releasing calcium from intracellular sources. ATP did not permeabilize keratinocytes, nor did it promote Ca influx into the cells. The half-maximal effect of ATP was at 10 microM, and saturation was observed at 30-100 microM. UTP, ITP, and ATP gamma S were as effective as ATP in releasing Cai from intracellular stores and competed with ATP for their response, whereas AMP and adenosine were ineffective, suggesting the specificity of P2 purinergic receptors in mediating the ATP response in keratinocytes. Single cell measurements revealed heterogeneity in the calcium response to ATP. This heterogeneity did not appear to be due to differences in the initial Cai response but to subsequent removal of increased Cai by these cells. ATP inhibited terminal differentiation of keratinocytes as measured by [35S]methionine incorporation into cornified envelopes and modestly stimulated incorporation of [3H]thymidine into DNA. Chelation of Cai by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid reduced basal Cai, blocked the Cai response to ATP, inhibited the basal rate of DNA synthesis, and blocked the ATP-induced increase in DNA synthesis. We conclude that extracellular ATP may be an important physiological regulator of epidermal growth and differentiation acting via IP3 and Cai.

Adenosine Triphosphate↗