Linking the ubiquitin-proteasome pathway to chromatin remodeling/modification by nuclear receptors.

Over 25 years ago, eukaryotic cells were shown to contain a highly specific system for the selective degradation of short-lived proteins, this system is known as the ubiquitin-proteasome pathway. In this pathway, proteins are targeted for degradation by covalent modification by a small highly conserved protein named ubiquitin. Ubiquitin-mediated degradation of regulatory proteins plays an important role in numerous cell processes, including cell cycle progression, signal transduction and transcriptional regulation. Recent experiments have shown that the ubiquitin-proteasome pathway is also involved in nuclear hormone receptor (NR)-mediated transcriptional regulation. The idea that the ubiquitin-proteasome pathway is involved in NR-mediated transcription is strengthened by experiments showing that ubiquitin-proteasome components are recruited to NR target gene promoters. However, it is not clear how these components modulate NR-mediated chromatin remodeling and gene expression. In this review, we postulate the role of the ubiquitin-proteasome pathway on NR-mediated chromatin remodeling and gene regulation based on the current knowledge from studies implicating the pathway in chromatin structure modifications that are applicable to NR function. Since evidence from this laboratory, using the glucocorticoid receptor responsive mouse mammary tumor virus (MMTV) promoter organized as chromatin, suggest that the ubiquitin-proteasome system may be involved in the elongation phase of transcription, we particularly concentrate on chromatin modifications associated with the elongation phase.

Association between Vitamin D receptor polymorphisms and the rate of bone loss in elderly women-importance of adjusting for dietary and lifestyle factors.

The association between the restriction length polymorphisms of the Vitamin D receptor (VDR) gene and the bone mineral density (BMD) or the rate of bone loss is still under debate. In a longitudinal study of untreated postmenopausal elderly women, we evaluated the relationship between the VDR gene polymorphisms (BsmI, TaqI, ApaI, and FokI) and the rate of bone loss over a 3-year period. We also examined the effect of adjustments for dietary and lifestyle factors on these associations. Before adjustments, the rate of femoral neck bone loss was - 3.76 +/- 1.58% in women with BB genotype and 0.45 +/- 0.65% in women with bb genotype, which was not significantly different. Upon adjustment for dietary and lifestyle factors, statistically significant (P = 0.03) bone loss was observed at femoral neck in women with BB genotype (- 3.66 +/- 2.44%) compared to that of bb genotype (2.39 +/- 1.32%). Similar results were observed with TaqI genotypes. The rates of bone loss at other skeletal sites were not different between VDR genotypes defined by BsmI and TaqI. VDR gene polymorphisms defined by ApaI and FokI were not related to the rate of bone loss.

Caffeine intake increases the rate of bone loss in elderly women and interacts with vitamin D receptor genotypes.

BACKGROUND: The role of caffeine as a risk factor for bone loss is controversial. OBJECTIVE: Our goals were 1) to compare in both a cross-sectional study and a 3-y longitudinal study the bone mineral density (BMD) of postmenopausal women consuming high or low amounts of caffeine and 2) to study the interaction between caffeine intake, vitamin D receptor (VDR) polymorphism, and BMD in the longitudinal study. DESIGN: The results are derived from cross-sectional measurements of BMD in 489 elderly women (aged 65-77 y) and from longitudinal measurements made in 96 of these women who were treated with a placebo for 3 y. Changes in BMD were adjusted for confounding factors and were compared between groups with either low (< or =300 mg/d) or high (>300 mg/d) caffeine intakes and between the VDR genotype subgroups of the low- and high-caffeine groups. RESULTS: Women with high caffeine intakes had significantly higher rates of bone loss at the spine than did those with low intakes (-1.90 +/- 0.97% compared with 1.19 +/- 1.08%; P = 0.038). When the data were analyzed according to VDR genotype and caffeine intake, women with the tt genotype had significantly (P = 0.054) higher rates of bone loss at the spine (-8.14 +/- 2.62%) than did women with the TT genotype (-0.34 +/- 1.42%) when their caffeine intake was >300 mg/d. CONCLUSIONS: Intakes of caffeine in amounts >300 mg/d ( approximately 514 g, or 18 oz, brewed coffee) accelerate bone loss at the spine in elderly postmenopausal women. Furthermore, women with the tt genetic variant of VDR appear to be at a greater risk for this deleterious effect of caffeine on bone.

The mouse mammary tumor virus promoter adopts distinct chromatin structures in human breast cancer cells with and without glucocorticoid receptor.

Steroid receptors represent a class of transcription regulators that act in part by overcoming the often repressive nature of chromatin to modulate gene activity. The mouse mammary tumor virus (MMTV) promoter is a useful model for studying transcriptional regulation by steroid hormone receptors in the context of chromatin. The chromatin architecture of the promoter prevents the assembly of basal transcription machinery and binding of ubiquitous transcription factors. However, in human breast carcinoma T47D cells lacking the glucocorticoid receptor (GR), but expressing the progesterone receptor (PR), nucleosome B (nuc B) assumes a constitutively hypersensitive chromatin structure. This correlation led us to test the hypothesis that the chromatin structure of nuc B was dependent on GR expression in T47D cells. To examine this possibility, we stably co-transfected the MMTV promoter and the GR into T47D cells that lacked both the GR and the PR. We found that in T47D cells that lack both the GR and the PR or express only the GR, nuc B assumes a constitutively "open" chromatin structure, which allows hormone independent access by restriction endonucleases and transcription factors. These results suggest that in GR(+)/pr(-) T47D cells, the MMTV chromatin structure permits GR transcriptional activation, independent of chromatin remodeling.

Selective activation of the glucocorticoid receptor by steroid antagonists in human breast cancer and osteosarcoma cells.

Steroid hormones regulate the transcription of numerous genes via high affinity receptors that act in concert with chromatin remodeling complexes, coactivators and corepressors. We have compared the activities of a variety of glucocorticoid receptor (GR) antagonists in breast cancer and osteosarcoma cell lines engineered to stably maintain the mouse mammary tumor virus promoter. In both cell types, GR activation by dexamethasone occurs via the disruption of mouse mammary tumor virus chromatin structure and the recruitment of receptor coactivator proteins. However, when challenged with a variety of antagonists the GR displays differential ability to activate transcription within the two cell types. For the breast cancer cells, the antagonists fail to activate the promoter and do not promote the association of the GR with either remodeling or coactivator proteins. In contrast, in osteosarcoma cells, the antiglucocorticoids, RU486 and RU43044, exhibit partial agonist activity. The capacity of these antagonists to stimulate transcription in the osteosarcoma cells is reflected in the ability of the RU486-bound receptor to remodel chromatin and associate with chromatin-remodeling proteins. Similarly, the observation that the RU486-bound receptor does not fully activate transcription is consistent with its inability to recruit receptor coactivator proteins.

Calciotropic hormones and bone markers in the elderly.

There is a lack of substantial data on changes in calciotropic hormones and bone markers in elderly subjects living in North America. Parathyroid hormone (PTH), serum 25-hydroxyvitamin D (25(OH)D) and bone markers (serum osteocalcin and urine N-telopeptide), were measured in 735 Caucasian subjects (235 men and 500 women) aged 65-87 years. There was a significant increase in serum osteocalcin and urine N-telopeptide with age in men, and a significant increase in serum osteocalcin with age in women. Serum PTH and 25(OH)D showed no significant change with age in men or women. After adjusting for age, calcium intake, serum creatinine, season, and weight, mean serum PTH (p = 0.01), serum osteocalcin (p = 0.0001) and 24 h urine N-telopeptide (p = 0.0001) were higher in women than men, and mean serum 25(OH)D (p = 0.0001) and 24 h urine calcium (p = 0.0001) were higher in men than women. Serum PTH was correlated with serum osteocalcin in men and women, r = 0.24, r = 0.17, p < 0.001, but not with urine N-telopeptide. Serum PTH was inversely correlated with serum 25(OH)D (r = -0.25, r = -034,p < 0.001), and positively correlated with serum creatinine (r = 0.14, r = 0.17,p < 0.01) in men and women. The prevalence of serum 25(OH)D levels below 12 ng/ml was only 33% in females and 0.4% in men. Thus vitamin D deficiency was very uncommon in the U.S.A. compared with Europe. Although mean serum PTH was increased in the elderly, only 4-6% had PTH levels above the normal range. In summary, the increase in serum PTH in the elderly can be explained more by changes in vitamin D status than by declining renal function. These data also show significantly higher (p = 0.001) bone remodeling markers in women.

Dietary calcium and vitamin D intake in elderly women: effect on serum parathyroid hormone and vitamin D metabolites.

In this study, the effect of dietary calcium and vitamin D on serum parathyroid hormone and vitamin D metabolites was measured in 376 free-living women aged 65-77 y. Mean calcium intake in both groups was close to the recommended dietary allowance of 800 mg/d. Mean vitamin D intake in the 245 women not taking vitamin D supplements was 3.53 microg/d (141 IU/d), which is below the recommended dietary allowance of 5 microg/d (200 IU/d). To test the hypothesis that vitamin D is more important than calcium in reducing serum parathyroid hormone, the source of dietary calcium intake was subdivided into milk, which is fortified with vitamin D, and nonmilk sources. The serum parathyroid hormone concentration was inversely correlated with calcium intake derived from milk (r = -0.20, P < 0.01) but not from nonmilk sources (r = -0.06). Furthermore, serum calcidiol correlated with milk calcium intake (r = 0.35, P < 0.001) but not with nonmilk calcium intake (r = 0.10). Multivariate analysis showed a significant effect of season on serum calcidiol but not on serum parathyroid hormone. Serum parathyroid hormone was inversely correlated with serum calcidiol (r = -0.33, P < 0.001) and the regression predicted that mean serum parathyroid hormone would be reduced in the elderly to concentrations considered normal in the young when serum calcidiol is 122 nmol/L (49 ng/mL); this would require a much higher recommended dietary allowance for vitamin D than 5 microg/d (200 IU/d).

Effect of vitamin D receptor genotypes on calcium absorption, duodenal vitamin D receptor concentration, and serum 1,25 dihydroxyvitamin D levels in normal women.

It is well established that bone mineral density is under strong genetic control. Recently it was reported that the Bsm I restriction fragment length polymorphism of the vitamin D receptor (VDR) gene could account for up to 75% of the genetic variance in bone mineral density. However, the physiological basis for such an effect has not been established. The VDR gene codes for the vitamin D receptor protein which regulates intestinal calcium absorption. In order to assess the biochemical basis we studied the effect of common allelic variation of the VDR gene on intestinal VDR protein concentration, calcium absorption, and serum 1,25 dihydroxyvitamin D (1,25(OH)2D). Ninety-two Caucasian women were genotyped for Bsm I and Taq I polymorphism at the VDR gene locus. From these we compared 49 young women aged 25-35 years and 43 elderly women aged 65-83 years, who had all three measurements performed. There were no significant differences in intestinal VDR protein concentration, serum 1, 25(OH)2D, or radioactive calcium absorption among VDR genotype groups. Therefore, the small intestine does not seem to be a target for VDR gene polymorphism.

Association between intestinal vitamin D receptor, calcium absorption, and serum 1,25 dihydroxyvitamin D in normal young and elderly women.

The exact mechanism for the decrease in intestinal calcium absorption with age is not yet understood. A decrease with age in serum 1,25-dihydroxyvitamin D (1,25(OH)2D) or a decrease in the intestinal vitamin D receptor (VDR) protein concentration are possible causes. The objective of this study was to examine the effect of age on these factors. Fifty-nine young women age 25-35 years were compared with 41 elderly women age 65-83 years who underwent measurements of VDR, calcium absorption using a 20 mg and 100 mg calcium carrier, and calciotropic hormones. Calcium absorption by both tests was lower in the elderly women compared with the young women (p < 0.05). Serum 1,25(OH)2D and duodenal VDR protein concentration were not significantly different between the two age groups. Serum 1,25(OH)2D correlated with the 20 mg calcium absorption test in both young (r = 0.35, p < 0.007) and elderly women (r = 0.58, p < 0.0001) and with the 100 mg calcium absorption in the elderly (r = 0.32; p < 0.05). VDR did not correlate with calcium absorption in young women or elderly women, nor did VDR correlate with serum 1,25(OH)2D and serum 25-hydroxyvitamin D. In summary, the decrease in calcium absorption cannot be explained by a decrease in intestinal VDR. The correlation between serum 1,25(OH)2D and both calcium absorption tests only accounts for 12-30% of the variance in the age-related change in the calcium absorption tests. Other factors, not yet understood, are responsible for the decline in calcium absorption with age.

Serum vitamin D metabolites and calcium absorption in normal young and elderly free-living women and in women living in nursing homes.

Vitamin D deficiency, which causes osteomalacia, may also be important in the pathogenesis of age-related osteoporosis. We studied serum vitamin D metabolites in 52 young women (mean age: 30 +/- 3 y; range: 25-35 y), 64 elderly free-living women (mean age: 71 +/- 4 y; range: 65-82 y), and 60 elderly women living in nursing homes (mean age: 84 +/- 9 y; range: 61-102 y). Mean serum 25-hydroxyvitamin D (calcidiol) was 10.8 +/- 4.4 nmol/L (27 +/- 11 ng/mL) in women living in nursing homes and was similar to that of free-living young (11.3 +/- 4.2 nmol/L, or 28 +/- 10 ng/mL) and elderly (11.5 +/- 3.2 nmol/L, or 29 +/- 8 ng/mL) women. Vitamin D deficiency (defined as serum calcidiol < 4.8 nmol/L, or 12 ng/mL) occurred in 8% of women living in nursing homes, in 6% of the young women, and in 1.6% of the free-living elderly women. Serum calcidiol was significantly correlated with vitamin D intake (r = 0.25, P < 0.05) and inversely correlated with serum intact parathyroid hormone (iPTH) (r = -0.16, P < 0.03). Serum iPTH increased with age and secondary hyperparathyroidism was observed in 17% of the women living in nursing homes. Calcium absorption declined with age, but calcium absorption and serum 1 alpha,25-dihydroxyvitamin D (calcitriol) were significantly lower in women living in nursing homes, which probably contributed to the secondary hyperparathyroidism. In conclusion, normal serum calcidiol may avoid the problem of osteomalacia, but it does not correct malabsorption of calcium. Although calcitriol corrects the malabsorption of calcium, it remains to be seen whether higher amounts of vitamin D can normalize the calcium malabsorption of aging.

Effect of parathyroid hormone (hPTH[1-34]) infusion on serum 1,25-dihydroxyvitamin D and parathyroid hormone in normal women.

Calcium absorption declines with age. Because 1,25-dihydroxyvitamin D (1,25(OH)2D) is the major hormone controlling calcium absorption, changes in vitamin D metabolism may account for the malabsorption of aging. Serum levels of 1,25(OH)2D have been reported to either decrease or remain unchanged with age. To assess the effect of aging on renal production of 1,25(OH)2D, we evaluated the response of renal 25OHD 1 alpha hydroxylase to human parathyroid hormone (hPTH(1-34) stimulation in 119 women ages 25-83 years. In this population, baseline serum 25OHD and 1,25(OH)2D values did not significantly change with age, but serum iPTH (r = 0.44; p < 0.001) and serum creatinine (r = 0.31; p < 0.01) increased with age. However, the stimulatory activity of hPTH(1-34) on the renal production of 1,25(OH)2D declined with age (r3 = -0.36; p < 0.001) and was most apparent after age 75, being 50% less than that of younger women. Besides age, the production of 1,25(OH)2D was found to be dependent on baseline serum iPTH (r = -0.31; p < 0.0001). Administration of hPTH(1-34) led to suppression of endogenous PTH, and suppressibility of endogenous PTH declined with age (r = 0.53; p < 0.0001). The increase in serum PTH and decreased suppressibility of PTH with age could be due to mild secondary hyperparathyroidism. The increase in PTH with age is probably responsible for maintaining normal serum 1,25(OH)2D levels in elderly subjects; however, decreased metabolism of 1,25(OH)2D in the elderly could also maintain normal serum 1,25(OH)2D levels.

Energy and protein metabolism of the Chinese pig.

Sixteen Chinese pigs (Meishan breed) from four litter outcome groups with initial weights ranging from 6 to 22 kg BW were used in a 28-d comparative slaughter experiment to determine the utilization of energy for maintenance and growth and the utilization of protein and amino acids (AA) by the Chinese pigs. Pigs were randomly allotted from litter outcome groups to four replicates of four pens each and to four treatments within replicates. A corn-soybean meal basal diet was formulated to provide all the nutrients except energy at twice the recommended levels for 10- to 20-kg pigs. The treatments were the basal diet fed at 3, 4, or 5% of BW. The fourth treatment was the initial slaughter group. As level of feeding increased, ADG increased linearly (P < .01) and gain:feed ratios increased quadratically (P < .06). Increasing the level of feeding had no effect on apparent digestibility coefficients of DM, N, or GE. Fecal N, urinary N, urinary urea N, and N retention increased linearly (P < .01) as feed intake increased. Level of feeding did not affect the DE, ME, or NE concentration in the diet. Metabolizable energy as a percentage of DE averaged 92.7% and was not affected by feeding level. Percentage of ether extract and DM of the empty body increased linearly (P < .01), but percentage of water decreased linearly (P < .01) with the level of feeding. Percentage of CP and ash of the empty body were not affected by the level of feeding (P > .05).(ABSTRACT TRUNCATED AT 250 WORDS)