Biomedical subjects

N J Koszewski

Publications and source records attributed to N J Koszewski.

At least 19 recordsLinked to original sources

PubMed2005

Contrasting mammalian PTH promoters: identification of transcription factors controlling PTH gene expression.

Parathyroid hormone (PTH) is a key component in the maintenance of calcium and phosphate homeostasis. The steady-state expression of the PTH gene can be modeled as a balance between transcriptional activators and repressors. During renal failure, the gradual loss of kidney function is often accompanied by increased circulating concentrations of PTH and decreased synthesis of 1,25-di-hydroxyvitamin D3 (1,25(OH)2D3). The latter finding results in impaired calcium absorption and the removal of a known repressor of PTH gene transcription. Current regimens for treating secondary hyperparathyroidism associated with renal insufficiency are focused on boosting activities that repress PTH gene transcription or secretion of the hormone, and involve the use of vitamin D and its analogues or calcimimetic agents. However, in recent years, concerns have arisen over the use of the steroid hormone and alternative treatments are being sought. Here, we present new information regarding transcription factors controlling PTH gene expression, which include the specificity proteins (Sp) and the nuclear factor Y (NF-Y) complex. A highly conserved DNA response element for the Sp proteins has been identified in mammalian promoters, while an NF-Y binding site is uniquely positioned in the human promoter. Both of these factors are expressed in the parathyroid gland and their DNA elements appear to be functioning as activators of PTH gene expression. Further elucidation of such pathways may offer novel approaches for treating hyperparathyroidism associated with renal failure via suppression of transcriptional activators. That work is currently in progress.

Gene Expression Regulation↗

PubMed2001

Isolation of genomic DNA sequences that bind vitamin D receptor complexes.

Vitamin D signaling is believed to be transduced by a heterodimeric receptor complex that binds to specific sequences of DNA termed vitamin D response elements (VDREs) in the promoter regions of target genes. However, recent studies have suggested that considerable flexibility exists in the types of binding sites the vitamin D receptor (VDR) is capable of recognizing, including some that bind VDR homodimers. In this report, a screening method involving immunoselection and PCR amplification was utilized to examine genomic binding sites for the receptor. Four individual fragments ranging in size from ca. 250-320 bp were nominally isolated from the amplified pool of captured fragments for further analysis. Each of the four sequences was capable of forming specific, unique VDR complexes using recombinant human VDR (rhVDR) alone or rhVDR heteromers formed in conjunction with the addition of recombinant human retinoid X receptor alpha (rhRXRalpha). Two of these fragments exhibited significant hormone-dependent repression of luciferase activity when linked to a thymidine kinase driven reporter vector. DNaseI footprinting revealed specific binding over DR+3 or related half-site sequences found within both of these DNA fragments. The results from this study demonstrate that specific, functional binding sites for the VDR can be successfully isolated from genomic DNA and should aid in the discovery of genes regulated by the steroid hormone.

Animals↗

PubMed2001

Evidence of functional vitamin D receptors in rat hippocampus.

The steroid hormone vitamin D has important biological roles in calcium transport, cell growth, and cell differentiation. Its cellular activities are mediated by high affinity interaction with the vitamin D receptor. In brain, autoradiographic, immunohistologic, and messenger RNA expression studies implicate a number of neuronal systems, including the hippocampus, as potential targets of vitamin D. However, cellular distribution and protein expression, and binding of the receptor to vitamin D response elements have yet to be established in hippocampus. This investigation was undertaken to characterize the vitamin D receptor in rat hippocampus with western blot, immunocytochemistry, and gel shift analyses. The presence of the receptor protein in hippocampus extracts was revealed with western blotting using an anti-rat vitamin D receptor antiserum. In vivo and in vitro immunocytochemical results confirmed the presence of vitamin D receptor in neuronal and glial cells. In the hippocampus, the receptor was localized in pyramidal and granule cell layers, CA1, CA2, and CA3 subfields and in the dentate gyrus. Double labeling for the vitamin D receptor and glial fibrillary acidic protein revealed that glia also expressed the receptor protein. Gel shift analyses evaluated with the murine osteopontin vitamin D response element indicated a specific, bound receptor-containing complex from hippocampal extracts. Altogether, these findings clearly document the localization of vitamin D receptor in rat hippocampus and that hippocampus contains vitamin D receptors capable of specifically binding to DNA. In combination with reports of a neuroprotective role for vitamin D in hippocampal cell survival, these data suggest that the endogenous vitamin D receptor may mitigate processes related to cellular homeostasis, perhaps through a calcium buffering mechanism.

Animals↗

PubMed2001

Parathyroid hormone/parathyroid hormone-related peptide type 1 receptor in human bone.

The parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor (denoted as PTH-1R) is a key signaling factor through which calcium-regulating hormones PTH and PTHrP exert their effects on bone. There are contradictory reports regarding the capability of osteoclasts to express PTH-1R. To address this issue in humans, bone biopsy specimen samples from 9 normal controls and 16 patients with moderate to severe secondary renal hyperparathyroid bone disease (2 degrees HPT) with elevated PTH levels were studied to determine whether osteoclasts in the bone microenvironment express PTH-1R messenger RNA (mRNA) and protein. We report that osteoclasts express the PTH-1R mRNA but the protein is detected only in patients with 2 degrees HPT. The PTH-1R mRNA and protein also were found in osteoblasts, osteocytes, and bone marrow cells. Receptor expression was higher in osteoclasts and osteoblasts of patients with 2 degrees HPT than normal controls (98.0 +/- 1.1% vs. 65.7 +/- 14.3% and 65.8 +/- 3.4% vs. 39.1 +/- 6.2%; p < 0.01, respectively). Approximately half of osteoclasts found in bone of patients with 2 degrees HPT have the PTH-1R protein. In patients with 2 degrees HPT, a positive relationship exists between erosion depth, a parameter of osteoclastic activity, and the percentage of osteoclasts with PTH-1R protein (r = 0.58; p < 0.05). In normal controls, an inverse relationship exists between the percentage of osteoblasts with receptor mRNA, mRNA signals/cell, and serum PTH levels (r = -0.82 and p < 0.05 and r = -0.78 and p < 0.01, respectively). The results provide the novel evidence of PTH-1R in human osteoclasts and suggest a functional role for the receptors in 2 degrees HPT.

Adult↗

PubMed2000

Vitamin D receptor interactions with positive and negative DNA response elements: an interference footprint comparison.

Interference footprinting protocols were utilized to examine the interactions of the vitamin D receptor (VDR) with either a positive or a negative vitamin D response element (VDRE). A sequence from the human osteocalcin (hOC) gene was chosen for the prototypical positive DR+3 VDRE, while an analogous sequence linked to the avian parathyroid hormone gene (aPTH) was used as the negative VDRE. Both types of response elements were examined for phosphate backbone contacts, as well as base-specific interactions with guanine and thymine residues. Sources of VDR included partially purified canine intestinal preparations, as well as extracts of recombinant human VDR and retinoid X receptor alpha prepared from baculovirus-infected Sf9 insect cells. Cold competition experiments using variable amounts of these oligonucleotides in the mobility shift assay revealed that the hOC element was a five-fold better competitor for heterodimer complex binding than the negative VDRE. Interference footprints revealed extensive strong contacts to the phosphate backbone and individual guanine and thymine nucleotides of the hOC element. The composite hOC footprint was asymmetric for the number and strength of interactions observed over each of the respective direct repeat half-sites. In contrast, the aPTH VDRE footprints revealed fewer points of DNA contact that were limited to the hexanucleotide repeat regions and were strikingly weaker in nature. The alignment of DNA contact points for both elements produced a 5' stagger that was indicative of successive major groove interactions, and consistent with dimer binding. DNA helical representations indicate that the heterodimer contacts to these response elements are substantially different and provide insight into functional aspects of each complex.

Animals↗

PubMed2000

Characterization of vitamin D receptor immunoreactivity in human bone cells.

The present study examined the expression of the vitamin D receptor (VDR) in adult human bone by immunohistochemical analysis. Antiserum from goats immunized with an N-terminal rat VDR peptide was purified by affinity chromatography. The purified antiserum recognized both endogenous rat and recombinant human VDR in Western blots. The purified antiserum was also able to specifically supershift the recombinant human VDR when analyzed in mobility shift assays. Immunohistochemical analysis of MG-63 cells, a human osteoblastic cell line known to express the VDR, revealed prominent staining over the nuclei of these cells. Immunostaining was greatly attenuated in the presence of an excess of the immunizing peptide. Analysis of bone biopsy samples from 16 normal human subjects immunostained for VDR protein showed strong, immunopositive staining over bone cells, particularly osteoblasts, in keeping with prior studies. In addition, there was significant immunoreactivity observed in nuclei of osteoclasts, lining cells and scattered bone marrow stromal cells of the adult human bone. Results showed that 298 osteoblasts out of 808 (36.9%) examined were immunopositive. It was also observed that 29 osteoclasts out of 125 (23%) contained VDR immunoreactivity. The ability to detect VDR in osteoclasts and stromal cell populations suggests that in addition to regulating osteoblast function, these other cell types are also direct targets of the hormone's action. These results demonstrate the utility of this purified antiserum in detecting the VDR in a variety of molecular techniques and should prove useful in examining receptor expression in various pathological conditions.

Adult↗

PubMed1999

1,25-Dihydroxyvitamin D(3) and 9-cis-retinoids are synergistic regulators of 24-hydroxylase activity in the rat and 1, 25-dihydroxyvitamin D(3) alters retinoic acid metabolism in vivo.

The RXR forms a heterodimer with the VDR to activate genes that are regulated by 1,25(OH)(2)D(3). In the absence of RXR's ligand, 9-cis-RA, RXR appears to be a silent partner to VDR. The effect of 9-cis-RA on VDR/RXR heterodimer formation and 1, 25(OH)(2)D(3)-mediated gene expression in vivo remains unclear. We examined the effect of exogenous 9-cis-RA or 9-cis-RA precursors, 9, 13-di-cis-RA and 9-cis-RCHO, on 1,25(OH)(2)D(3)-mediated induction rat renal 24-hydroxylase. The rats were treated as follows: (1) vehicle; (2) 1,25(OH)(2)D(3); (3) 1,25(OH)(2)D(3) + 9-cis-RA; (4) 1, 25(OH)(2)D(3) + 9,13-di-cis-RA; (5) 1,25(OH)(2)D(3) + 9-cis-RCHO; (6) 9-cis-RA; (7) 9,13-di-cis-RA; and (8) 9-cis-RCHO. 1, 25(OH)(2)D(3) was administered IP 18 h prior to sacrifice. The retinoids were administered every 4 h, starting 28 h prior to sacrifice. The last retinoid dose was administered 4 h prior to sacrifice. Treatment with 1,25(OH)(2)D(3) alone increased 24-hydroxylase from 35 +/- 6 (controls) to 258 +/- 44 pmol/min/g tissue. When 1,25(OH)(2)D(3) was administered with 9-cis-RA, 9, 13-di-cis-RA, or 9-cis-RCHO, 24-hydroxylases were 568 +/- 56, 524 +/- 56, and 463 +/- 62 pmol/min/g tissue, respectively. Furthermore, codosing of 1,25(OH)(2)D(3) and 9-cis-retinoids resulted in higher circulating concentrations of 9-cis-RA and 9,13-di-cis-RA when compared to rats dosed with 9-cis-retinoids alone. This was shown to be due to 1,25(OH)(2)D(3) increasing the half-life of 9,13-di-cis-RA by three to four times. These results show that 9-cis-RA can act synergistically with 1,25(OH)(2)D(3) in the regulation of 24-hydroxylase in vivo. Additionally, 1,25(OH)(2)D(3) regulates 9, 13-di-cis-RA metabolism in vivo.

Animals↗

PubMed1999

Turning a negative into a positive: vitamin D receptor interactions with the avian parathyroid hormone response element.

1 ,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] negatively regulates expression of the avian PTH (aPTH) gene transcript, and a vitamin D response element (VDRE) near the promoter of the aPTH gene had previously been identified. The present report assessed whether the negative activity imparted by the aPTH VDRE could be converted to a positive transcriptional response through selective mutations introduced into the element. The tested sequences were derived from individual and combined mutations to 2 bp in the 3'-half of the direct repeat element, GGGTCAggaGGGTGT. Cold competition experiments using mutant and wild-type oligonucleotides in the mobility shift assay revealed minor differences in the ability of any of these sequences to compete for binding to a heterodimer complex comprised of recombinant proteins. Ethylation interference footprint analysis for each of the mutants produced unique patterns over the 3'-half-sites that were distinct from the weak, wild-type footprint. Transcriptional outcomes evaluated from a chloramphenicol acetyltransferase reporter construct utilizing the aPTH promoter found that the individual T-->A mutant produced an attenuated negative transcriptional response while the G-->C mutant resulted in a reproducibly weak positive transcriptional outcome. The double mutant, however, yielded a 4-fold increase in transcription, similar to the 7-fold increase observed from an analogous construct using the human osteocalcin VDRE. UV light crosslinking to gapped oligonucleotides assessed the polarity of heterodimer binding to the wild-type and double mutant sequences and was consistent with the vitamin D receptor preferentially binding to the 5'-half of both elements. Finally, DNA affinity chromatography was used to immobilize heterodimer complexes bound to the wild-type and double mutant sequences as bait to identify proteins that may preferentially interact with these DNA-bound heterodimers. This analysis revealed the presence of a p160 protein that specifically interacted with the heterodimer bound to the wild-type VDRE, but was absent from complexes bound to response elements associated with positive transcriptional activity. Thus, the sequence of the individual VDRE appears to play an active role in dictating transcriptional responses that may be mediated by altering the ability of a vitamin D receptor heterodimer to interact with accessory factor proteins.

Animals↗

PubMed1999

Vitamin D receptor interactions with the rat parathyroid hormone gene: synergistic effects between two negative vitamin D response elements.

Vitamin D response elements (VDREs) that are required for negative regulation of rat parathyroid hormone (rPTH) gene expression have been characterized. Gel mobility shift assays using DNA restriction enzyme fragments and recombinant proteins for vitamin D and retinoic acid X receptors (VDR/RXR) revealed a sequence between -793 and -779 that bound a VDR/RXR heterodimer with high affinity (VDRE1). Furthermore, a lower affinity site (VDRE2) was detected that acted in combination with VDRE1 to bind a second VDR/RXR complex. As determined by ethylation interference analysis, the nucleotide sequence of VDRE1 consisted of GGTTCA GTG AGGTAC, which is remarkably similar to the sequence of the negative VDRE found in the chicken PTH (cPTH) gene. Using the same technique, VDRE2 was identified between positions -760 and -746 and contained the sequence AGGCTA GCC AGTTCA. Functional analysis was determined by transfection studies with plasmid constructs that expressed the gene for chloramphenicol acetyl transferase (CAT). The ability of the VDREs to regulate gene expression was tested in their native context with the rPTH promoter as well as when positioned immediately upstream from the cPTH promoter. With either plasmid construct, exposure to 10(-8)M 1,25(OH)2D3 resulted in a 60-70% decrease in CAT gene expression when both VDRE1 and VDRE2 were present. Examination of the individual VDREs showed that inhibition by 10(-8) M 1,25(OH)2D3 was only 35-40% when just VDRE1 was present. By itself, VDRE2 was even less effective, as significant inhibition of CAT activity (20%) was observed only in the presence of higher concentrations of 1, 25(OH)2D3 (10(-7)M) or when a plasmid vector that overexpressed the VDR protein was cotransfected. In conclusion, the rPTH gene contains two negative VDREs that act in concert to bind two RXR/VDR heterodimer complexes and that both VDREs are required for maximal inhibition by 1,25(OH)2D3.

Animals↗

PubMed1999

Differential effects of 20-epi vitamin D analogs on the vitamin D receptor homodimer.

Vitamin D analogs have received increased attention because of their possible therapeutic benefits in treating osteoporosis and various proliferative disorders. Several analogs were examined for their effects on DNA binding of the vitamin D receptor (VDR) homodimer complex with the murine osteopontin vitamin D response element. All of the tested analogs increased complex binding by recombinant human VDR in the electrophoretic mobility shift assay and notable differences in mobility of these complexes were observed. A panel of C-terminal anti-VDR antisera were screened for their ability to interact with analog-bound VDR homodimer complexes or as a heterodimer complex with recombinant human retinoid X receptor alpha (rhRXR alpha). Like calcitriol, analog-bound heterodimer complexes were largely resistant to interaction with these antisera; however, striking differences were observed with the various antisera in an analogous homodimer binding experiment. KH1060 and CB1093, analogs with 20-epi conformations, produced homodimer complexes that were 3- to 6-fold more resistant to supershifting with Ab180 compared with the hormone or EB1089. Chymotrypsin digestion in combination with Western blotting using a C-terminal anti-VDR antiserum revealed similar digestion patterns for all ligands. However, KH1060- and CB1093-bound VDR complexes were more resistant to digestion than either calcitriol or EB1089. Finally, the ability of these compounds to yield stable homodimer complexes was assessed by challenging preformed homodimer with the exogenous addition of rhRXR alpha extracts. Although new heterodimer complexes appeared in a time-dependent fashion, the preformed homodimer complexes exhibited stable binding throughout the time course of the experiment. The results indicate that VDR homodimers are targets of vitamin D analogs with differential effects on C-terminal protein conformation that may partially explain the varied biological responses of these compounds.

Amino Acid Sequence↗

PubMed1998

Tuberous sclerosis gene 2 product modulates transcription mediated by steroid hormone receptor family members.

Tuberous sclerosis (TSC) is a genetic disorder that results in the development of hamartomatous lesions in a variety of organ systems. Both the prevalence of the disease and the often devastating consequences of these tumors pose a serious health and medical care problem. The disease has been mapped to two distinct genetic loci in humans, and although the genes (TSC1 and TSC2) for both loci have recently been cloned, their function remains an enigma. Data presented here demonstrates that TSC2 protein can bind and selectively modulate transcription mediated by members of the steroid receptor superfamily of genes. These data place TSC2 into a growing list of nuclear receptor coregulators and strengthen the expanding body of evidence that these coregulators may play critical roles in cellular differentiation.

Amino Acid Sequence↗

PubMed1998

Selectivity of a C-terminal peptide antiserum for different DNA-binding states of the vitamin D receptor.

Antisera against peptides from the extreme N- and C-terminal regions of the VDR were evaluated. The N-terminal antiserum Ab192 functioned as a general-purpose antibody, being able to supershift the rhVDR in heterodimeric or homodimeric binding complexes in the EMSA, and detect both recombinant and native forms of the receptor on Western blots. The C-terminal antiserum, Ab195, also identified the receptor on Western blots, but in contrast, it displayed differential sensitivity to the conditions employed in the EMSA. In the presence of 1,25(OH)2D3, rhVDR, rhRXR alpha, and nonspecific DNA, Ab195 produced a weak supershift of the heterodimer complex in the EMSA. Significantly, omission of hormone from the binding buffer resulted in a complete shift of the bound complex with the antiserum. A complete supershift was also observed if only the nonspecific DNA was removed. Together these results indicate antiserum sensitivity to the ligand status in the rhVDR C-terminus as part of a DNA-bound heterodimer complex. Inclusion of 9-cis RA resulted in a modest increase in the amount of shifted product relative to 1,25(OH)2D3 alone. Finally, Ab195 completely supershifted the rhVDR homodimer binding complex under all tested conditions, including those analogous to where it was largely ineffective in shifting the heterodimer. Thus, Ab195 is sensitive to the ligand binding status of the VDR, discriminates heterodimer and homodimer binding interactions, and should provide an additional tool for exploring conformational changes induced in the receptor.

Amino Acid Sequence↗

PubMed1998

Calreticulin inhibits vitamin D's action on the PTH gene in vitro and may prevent vitamin D's effect in vivo in hypocalcemic rats.

1,25-dihydroxyvitaminD3 [1,25-(OH)2D3] and PTH both act to increase serum calcium. In addition, 1,25-(OH)2D3 decreases PTH gene transcription, which is relevant both to the physiology of calcium homeostasis and to the management of the secondary hyperparathyroidism of patients with chronic renal failure. In chronic hypocalcemia there is secondary hyperparathyroidism with increased levels of PTH mRNA and serum PTH despite markedly increased levels of 1,25-(OH)2D3. We have studied the role of calreticulin in this resistance to 1,25-(OH)2D3. Weanling rats fed a low-calcium diet were hypocalcemic and had increased PTH mRNA levels despite high serum 1,25-(OH)2D3 levels. 1,25-(OH)2D3 given by continuous minipump infusion to normal rats led to the expected decrease in PTH mRNA. The hypocalcemic rats had an increased concentration of calreticulin in the nuclear fraction of their parathyroids, but not in other tissues. Gel shift assays showed that a purified vitamin D receptor and retinoid X receptor-beta bound to the PTH promoter's chicken and rat vitamin D response element (VDRE), and this binding was inhibited by added pure calreticulin. Transfection studies with a PTH VDRE-chloramphenicol acetyltransferase (CAT) construct showed that 1,25-(OH)2D3 decreased CAT transcription. Cotransfection of PTH VDRE-CAT with a calreticulin expression vector in the sense orientation prevented the transcriptional effect of 1,25-(OH)2D3, but a calreticulin vector in the antisense orientation had no effect. These results show that calreticulin prevents the binding of vitamin D receptor-retinoid X receptor-beta to the PTH VDRE in gel retardation assays and prevents the transcriptional effect of 1,25-(OH)2D3 on the PTH gene. This is the first report of calreticulin inhibiting a down-regulatory function of a sterol hormone and may help explain the refractoriness of the secondary hyperparathyroidism of many chronic renal failure patients to 1,25-(OH)2D3.

Animals↗

PubMed1997

Secondary hyperparathyroidism and vitamin D receptor binding to vitamin D response elements in rats with incipient renal failure.

The pathogenesis of secondary hyperparathyroidism in early renal failure is poorly understood. In the study presented here, parathyroid hormone and GFR in rats with mild renal failure of various durations are evaluated. Parathyroid hormone increased significantly 3 days after nephrectomy and peaked at 2 wk, despite reduction in GFR of < 50%. Parathyroid hormone remained elevated, but there was no difference in serum levels of calcium, phosphorus, and calcitriol between the nephrectomized and sham-operated rats. There were also no differences in both intestinal and kidney vitamin D receptor concentrations between the two groups. Histomorphometric analysis of bone at 6 wk revealed significant increase in osteoid thickness, osteoblast number, erosion surface with osteoclasts, and erosion depth. Employing electrophoretic mobility shift assay, we consistently observed a significant reduction in kidney calcitriol-receptor complex binding to mouse osteopontin vitamin D response element (-70.2 +/- 4.9%, P < 0.001). Western blot analysis also revealed a significant reduction in at least one retinoid X receptor isoform. In conclusion, biochemical and histological evidence of secondary hyperparathyroidism develops in rats with mild renal failure, despite normal calcium, phosphorus, calcitriol, and vitamin D receptor concentrations. These rats also have evidence of reduced renal vitamin D receptor binding to nuclear response elements. This finding, possibly an important early factor in the pathogenesis of secondary hyperparathyroidism, could also play a role in the development of compensatory renal growth of the remnant kidney.

Animals↗

PubMed1996

Vitamin D receptor interactions with the murine osteopontin response element.

The nature of the DNA binding interactions of the human vitamin D receptor (hVDR) with the murine osteopontin vitamin D response element (mOP VDRE) was examined. Both recombinant hVDR and human retinoid X receptor beta (hRXRbeta) proteins were obtained from baculovirus-infected Sf9 insect cells. Mixing extracts of the two recombinant proteins resulted in the strong, specific formation of a slower migrating complex in the electrophoretic mobility shift assay. Crude extracts of the expressed hVDR alone were also capable of binding with high affinity to the mOP sequence, and this binding was enhanced in the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Competition experiments confirmed the specificity of this interaction and revealed that the human osteocalcin VDRE was a poor competitor for this binding. Ethylation interference footprint analyses of hVDR/hRXRbeta and hVDR complexes revealed only subtle differences in how these two different VDR-containing complexes interacted with the mOP VDRE. The footprints displayed contact points in both halves of the direct repeat format, confirming the dimeric and major groove interactions of both types of complexes. DNA affinity chromatography of labelled hVDR extracts revealed a peak eluting at ca. 290 mM KC1 that was capable of rebinding to the mOP sequence in gel shift experiments. Ultraviolet (UV) light-crosslinking experiments of hVDR extracts alone to radiolabelled DNA were consistent with the existence of a homodimeric hVDR interaction. Additionally, these experiments confirmed the direct interaction of a hVDR/hRXRbeta heterodimer when mixed extracts were utilized. From these results we infer that homodimers of the hVDR which respond with enhanced DNA binding to particular vitamin D response elements when exposed to 1,25-(OH)2D3 are possible. This may be of functional significance when RXR proteins are limiting or RXR ligand is present within a cell.

Animals↗

PubMed1996

Bone resorption and mRNA expression of IL-6 and IL-6 receptor in patients with renal osteodystrophy.

The cytokine interleukin-6 (IL-6) is a major cell regulatory factor that may play an important role in the bone remodeling of patients with renal failure. IL-6 exerts its action by binding to its receptor (IL-6R), which leads to transduction of a second messenger cascade within cells. In vitro as well as in vivo data point to IL-6 as an autocrine/paracrine factor in bone osteoclasts. Recently, bone cells from patients with Paget's disease were found to express IL-6 and IL-6R mRNA transcripts. However, in patients with renal bone disease, there is currently no in vivo evidence that osteoclasts have the capability to express mRNA for IL-6 and IL-6R. To investigate the potential expression of IL-6 and IL-6R in bone and its relationship to bone cell activity, iliac crest bone biopsies were performed in patients on chronic maintenance dialysis. Messenger RNA expression of IL-6 and IL-6R was studied using in situ hybridization histochemistry, and parameters of bone turnover were determined by bone histomorphometry. In the samples studied, mRNA expression of IL-6 and IL-6R was found in osteoclasts and bone marrow cells. Furthermore, we report the novel finding of increased IL-6R mRNA expression in osteoclasts engaged in increased bone resorption. The results of the present study suggest that the cytokine IL-6 is intricately involved in osteoclastic bone resorption and that expression of its receptor, IL-6R, in osteoclasts may parallel osteoclastic bone resorbing activity.

Adult↗

PubMed1995

9,13-Di-cis-retinoic acid is the major circulating geometric isomer of retinoic acid in the periparturient period.

This study examines changes in the geometric isomers of retinoic acid (RA) during late gestation and early lactation. Blood samples were collected from dairy cows at 14 days prepartum and continuing until 14 days postpartum. Assays were done using solid-phase extraction followed by quantitation of RA isomers by reverse-phase HPLC. The major RA in plasma of periparturient cows was 9,13-di-cis-RA. 9,13-Di-cis-RA was present at < or = 1 ng/ml 10 days prior to parturition and was elevated to approximately 2 ng/ml by 1 day prepartum, then increased to > 4 ng/ml by 1 day postpartum. 9,13-Di-cis-RA remained at approximately 4 ng/ml for at least 14 days postpartum. Plasma all-trans-RA, 13-cis-RA, and 9-cis-RA were also elevated during the periparturient period with the most pronounced changes (approximately 25-30% increase) occurring between the day of parturition and 4 days postpartum. 9,13-Di-cis-RA was 116- and 30-fold less competitive than 9-cis-RA for binding to human retinoid X receptors (hRXR)alpha and hRXR beta, respectively. Because of this relatively low affinity for the RXR, it seems that 9,13-di-cis-RA per se would contribute marginally, if at all, to the pleiotropic effects attributed to 9-cis-RA. This study establishes that profound changes in vitamin A metabolism occur in late gestation and early lactation. Because 9,13-di-cis-RA is a metabolite of 9-cis-RA, these data imply that there are dramatic fluctuations in 9-cis-RA which are more profound than other physiologically occurring RAs, particularly all-trans-RA.

Animals↗

PubMed1993

Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor.

We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor.

Animals↗