K L Sunn

Publications (5)18.35 Total impact

• Article: Novel N-terminal variant of human VDR.

  K L Sunn, T A Cock, L A Crofts, J A Eisman, E M Gardiner
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  ABSTRACT: The importance of N-terminal regions of nuclear hormone receptors in transcriptional regulation is increasingly recognized. As variant VDR gene transcripts indicated possible N-terminally extended receptors, we investigated their natural occurrence, transactivation capacity, and subcellular localization. A novel 54-kDa VDRB1 protein, in addition to the previously recognized 48-kDa VDRA form, was detected in human kidney tissue as well as in osteoblastic (MG63), intestinal (Int-407, DLD-1, and COLO 206F), and kidney epithelial (786) human cell lines by Western blots using isoform-specific and nonselective anti-VDR antibodies. VDRB1 was present at approximately one-third the level of VDRA. Isoform-specific VDRB1 expression constructs produced lower ligand-dependent transactivation than VDRA when transiently transfected with a vitamin D-responsive promoter into cell lines with low endogenous VDR. Intracellular localization patterns of the green fluorescent protein-tagged VDR isoforms differed. VDRB1 appeared as discrete intranuclear foci in the absence of 1,25-dihydroxyvitamin D3, whereas VDRA produced diffuse nuclear fluorescence. After 1,25-dihydroxyvitamin D3 treatment, both VDR isoforms exhibited similar diffuse nuclear signal. In the absence of 1,25-dihydroxyvitamin D3, the VDRB1 foci partially colocalized with SC-35 speckles and a subset of promyelocytic leukemia nuclear bodies. These data provide the first evidence of VDRB1, a novel N-terminally variant human VDR that is expressed at a level comparable to VDRA in human tissue and cell lines. It is characterized by reduced transactivation activity and a ligand-responsive speckled intranuclear localization. The intranuclear compartmentalization and altered functional activity of VDRB1 may mediate a specialized physiological role for this receptor isoform.
  Molecular Endocrinology 10/2001; 15(9):1599-609. · 4.54 Impact Factor
• Article: Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage.

  E M Gardiner, P A Baldock, G P Thomas, N A Sims, N K Henderson, B Hollis, C P White, K L Sunn, N A Morrison, W R Walsh, J A Eisman
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  ABSTRACT: The microarchitecture of bone is regulated by complex interactions between the bone-forming and resorbing cells, and several compounds regulate both actions. For example, vitamin D, which is required for bone mineralization, also stimulates bone resorption. Transgenic mice overexpressing the vitamin D receptor solely in mature cells of the osteoblastic bone-forming lineage were generated to test the potential therapeutic value of shifting the balance of vitamin D activity in favor of bone formation. Cortical bone was 5% wider and 15% stronger in these mice due to a doubling of periosteal mineral apposition rate without altered body weight or calcium homeostatic hormone levels. A 20% increase in trabecular bone volume in transgenic vertebrae was also observed, unexpectedly associated with a 30% reduction in resorption surface rather than greater bone formation. These findings indicate anabolic vitamin D activity in bone and identify a previously unknown pathway from mature osteoblastic cells to inhibit osteoclastic bone resorption, counterbalancing the known stimulatory action through immature osteoblastic cells. A therapeutic approach that both stimulates cortical anabolic and inhibits trabecular resorptive pathways would be ideal for treatment of osteoporosis and other osteopenic disorders.
  The FASEB Journal 11/2000; 14(13):1908-16. · 5.71 Impact Factor
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  Available from: endocrinology-journals.org

  Article: Expression of the parathyroid Ca(2+)-sensing receptor in cytotrophoblasts from human term placenta.

  R A Bradbury, K L Sunn, M Crossley, M Bai, E M Brown, L Delbridge, A D Conigrave
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  ABSTRACT: Fura-2-loaded human cytotrophoblasts responded to elevated extracellular Ca2+ concentration ([Ca2+]o) with monophasic or, in the case of large (> 20 microns) extravillous cells, biphasic elevations in intracellular free Ca2+ ion concentration ([Ca2+]i) that returned to baseline levels after restoration of control [Ca2+]o. Large extravillous cytotrophoblasts also responded to elevated [Mg2+]o with transient elevations in [Ca2+]i, consistent with the behaviour of the parathyroid Ca2(+)-sensing receptor. Expression of the parathyroid Ca2(+)-sensing receptor in placental cells was confirmed using Northern blot and reverse transcription (RT)-PCR analysis. However, the major transcript in human placental cells (6.2 kb) differed from that expressed by human parathyroid cells (5.6 kb). RT-PCR analysis and DNA sequencing of key PCR products also revealed the presence of a splice variant in placental and parathyroid cells that lacks exon 3.
  Journal of Endocrinology 03/1998; 156(3):425-30. · 3.55 Impact Factor
• Article: Human and murine osteocalcin gene expression: conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo.

  N A Sims, C P White, K L Sunn, G P Thomas, M L Drummond, N A Morrison, J A Eisman, E M Gardiner
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  ABSTRACT: Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6+/-3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2+/-0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2+/-7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5+/-0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly upregulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.
  Molecular Endocrinology 11/1997; 11(11):1695-708. · 4.54 Impact Factor
• Article: Enhanced transactivation activity of vitamin D receptor B1 associated with focal nuclear accumulation and cofactor binding

  J L Flanagan, K L Sunn, G M Leong, C. Fong, A P Kouzmenko, J A Eisman, E M Gardiner
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  ABSTRACT: Biological responses to 1,25 dihydroxyvitamin D3 are mediated by the vitamin D receptor (VDR), of which there are two protein isoforms. The A/B region of the recently identified VDRB1 isoform is extended at the N-terminus by 50 amino acids producing a 73 amino acid A/B region, compared with the 23 amino acids of the conventional VDRA receptor. As A/B regions of nuclear receptors often contain ligand-independent activation function (AF-1) domains, the functional properties of this region of VDRB1 might differ from those of VDRA. The VDRB1 isoform exhibited greater (144% ± 17%) transactivation of a rat 1,25-dihydroxvitamin D-24-hydroxylase reporter construct than VDRA in transiently transfected COS-1 cells 6 hr post treatment with 10nM 1,25-dihydroxyvitamin D3. This difference was not apparent at 16 hr post treatment. Evidence for AF-1 activity in the VDRB1 N-terminal A/B region was observed in yeast and mammalian one-hybrid experiments. The VDRB1 A/B region exhibited strong AF-1 activity (8-fold ligand-independent activation), compared to the VDRA A/B region (3-fold activation). Similarly, there was stronger interaction between the A/B region of VDRB1 and the VDR ligand binding domain (LBD) in mammalian two-hybrid and GST pull-down assays (3-fold in the presence of ligand, whereas interaction between the VDRA A/B domain and the LBD was weak. Introduction of an E420Q mutation into the LBD, which inactivates C-terminal ligand-dependent transactivation (AF-2) activity by abolishing recruitment of coactivators, markedly reduced but did not abolish ligand-dependent interaction with the VDRB1 A/B domain. This suggests that, as well as cofactor mediated interaction between the VDRB1 A/B domain and the LBD, there may be a direct AF-1/AF-2 interaction. In the absence of ligand, GFP-tagged VDRB1 accumulated in discrete nuclear foci whereas VDRA exhibited a diffuse nuclear signal. VDRB1 foci were not observed with the E420Q mutation, suggesting that speckle formation may be mediated at least in part by A/B domain-LBD interaction or by cofactor recruitment in the absence of ligand. Ligand-independent interaction of p160 co-activator proteins with full-length wildtype VDRB1, but not with the E420Q mutant VDRB1 or wildtype VDRA in GST-pull-down assays, supports the latter concept. Thus, accumulation of VDRB1 in nuclear foci may relate to its differential binding to transcriptional co-activators in the absence of ligand and the physical association of cofactors in the absence of ligand may allow for a more rapid response to ligand leading to the more effective transactivation observed.