Elevated osteoblastic vitamin D receptor reduces calcium deficient bone loss in transgenic mice.

Abstracts

Bone Vol. 27, No. 3, Supplement: October 2000: 1Sp54S

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02 EXPRESSION OF RANK, RANKL AND OPG IS ASSOCIATED WITH RHEUMATOID ARTHRITIC, PERIODONTAL AND PERIPROSTHETIC TISSUES

TN Crotti I, M Loricl,

M. Dalidjanl,

GJ Atkins*,

DM Findlay2,

DW

Howe2 and DR Haynes’ Department of Pathology and Department of Orthopaedu and Trauma, University of Adelaide, SA, 5005 Australia. Bone lysis is characteristic of rheumatoid arthritis, periodontal disease, and aseptic loosening of prosthetic joints. Our previous reports show that elevated expression of osteoclastogenic stimulators, RANKL (also known as osteoclast differentiation factor) and its receptor RANK, and reduced expressIon of the inhibitor osteoprotegerin (OPG) correlates with the atxlity of cells in periprosthetx and rheumatoid tissues to develop into osteoclasts. The aim of this study was to investigate the cell types that express these regulators of osteoclast development in tissues near bone lysis. Cells were extracted from these tissues and osteoclastogenic mediator expression in viva was assessed using semi-quantitative reverse transcription polymerax chain reaction (RT-PCR). In situ hybrldisation using DIG labelled nboprobes specific for RANK mRNA was used to identify cells that might become osteoclasts. lmmunohistochemlstry using antibodies to CD68 was also used to identify monocytes and macrophages. RT-PCR showed that RANKL, RANK and OPG mRNA was expressed by cells present in per]-prosthetic revision tissues, rheumatoid tissues and periodontal tissue. The expression of OPG relatwe to RANKL was higher in non-diseased control tissues than In AlfU those wth periodontal disease and rheumatoid samples. hybrldlsation showed RANK expression by both CD68+ and CD68cells in all tissues. Many of the cells expressing RANK m the perlprosthetic tissues contained prssthetic particles. The results show that the relative levels of OPG and RANKL along wth RAYK exprcsslon may regulate osteoclastic activity m these common pathologies.

03 ELEVATED OSTEOBLASTIC VITAMIN D RECEPTOR REDUCES CALCIUM DEFICIENT BONE LOSS IN TRANSGENIC MICE PA Baldock, GP Thomas, NA Sims, NK Henderson B Hollis’, JA E~sman. EM Gardiner Bone and Mineral Research Program, C&van Institute of Medical Research; ‘Department of Pediatrics, Medical University of South Carolina, Charleston, SC Transgenic upregulation of vitamin D receptor (VDR) in mature osteoblastic cells resulted in stronger, wider long bones with elevated periosteal bone formation in a mouse model More abundant trabecular bone was associated with reduced ‘osteoclast surface, rather than altered bone formation These findings were obtained following a modest reductmn In dietary calcium intake fclr one month The present study was designed to examine any dependence of this phenotype on a calcium deficient state. Mice were maintained on 0 9% Ca chow diet, bone and calcium homeostatic parameters were analysed at 7 and X months of age Two lines of transgenic mice (OSV3 and 9) were collected and compared to non-transgenic FVB controls (meaniSE) Serum calciotropic parameters did not differ between genetic groups total calcium (mmofl), FVB 3 MO 1, OSV9 2 9;tO.l. 0SV3 2 X+0 1, PTH (ng/ml), FVB 8315, OSV9 8215. 0SV3 85k9, and 1,25(OH)2D3 (pmol/l), FVB 137+7, 0SV9 137110, 0SV3 139+12 Femoral cortical bone area was elevated (8%, P
BONE-SPECIFIC CONTROL OF VITAMIN D METABOLlSM PH Anderson, S lida, HA Morns, BK Ma), M Cochran Dept of Physiology & Blochenustv. Unwerslty of Adelaide. Adelaide Chmcal Btochermstry, IMVS, Adelade The current paradigm of renal activation of vmmun D (1.25D) by 25. h>drosywtamm D la-hydroxylase (CYPI). transport to target organs and breakdown by local 1,25-hydroxywtamm D 24.hydrosylase (CYP24) cannot explain many of the IYI vifro actlrities of 1,25D includmg those on the bone cells. Local 1,25D synthesis by bone cells has been proposed to explain the clinical paradox between the chmcal features of vItanun D msuffiaency such as that assouated with hip fractures, and normal circulating 1,25D levels We habe studled the relationshp belween 1,25D status and dietruy calcwm (Ca) levels on CYPl and CYP24 mRNA expression levels m both kidney and bone tissues Vitamin D-deplete rats were bred m an UV free environment and fed wtamm D-deficient diet contammg 1% Ca. Six female offsprmg were mamtamed on the same diet until 6 months of age. Vitamm Dreplete rats were fed either a Ca-deticlent diet or 2% Ca &et or standard (0 4% Ca) for 40 days until 6 months Femora and kidneys were removed from rats at 6 months Total RNA &as extracted from these tissues by phenol/chloroform methods. Messenger RNA was quantrlied by Real-Time PCR after reverse transcnption In bltamm D-deplete rats. CYPI mRNA expressIon mcreased 21-fold m the kldneq and 11 -fold m the bone ivhen compared with rats fed standard diet In addmon. a5-fold decrease in kidney and opposmg 2-fold stlmulatlon m bone CYPZJ mRNA le\ els w’as seen Rats fed a Ca-deficient diet demonstrated a 12. fold increase in renal CYPI expression and a modest 2-fold increase m the bone CYPl mRNA. Although kidney CYP24 levels were unaltered. there was a 5-fold decrease m bone CYP24 mRNA levels. Rats fed 2% Ca diet demonstrated a 5-fold decrease m renal CYPI expression m whde bone CYPI levels remained unchanged. CYP24 mRNA levels were suppressed between 4- and h-fold In both kidney and bone It IS clear from these Iindmgs that there is tissue-specific control of CYPI and CYP24 mRNA expressIon, which IS altered bv hormonal and dIetan factors. We have now demonstrated bone-specllfic control of vitamin i, metabohsm, which regulate the supply and clearance of I .25D from bone. cells durmg bone metabohsm

04 TDENTIFICATION OF PROTEIN WTERACTORS WITH PARATHYROID HORMONE-RELATED PROTEIN (PTHrP). L A Co&n. T J Martin and M T Gillespie St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy 3065, Victoria Parathyroid hormone-related protein is the causal factor for hypercalcaemia associated with malignancy (HHM) PTHrP acquires humoral characteristics, not observed in normal adults Since its discovery, its role in normal physiology has been pursued PTHrP has been shown to be involved in differentiation of chondrocytes in the developing embryo, smooth muscle relaxation, placental calcium transport and mammary gland development. Also, PTHrP expression and intracellular localisation is tightly associated with the cell cycle This suggests that PTHrP may have intracellular functions distinct from those elicited as a consequence of cell surface signalling We have attempted to identify protein interactors with PTHrP using the yeast two-hybrid system, in order to define proteins important for the intracellular localisation of PTHrP as well as those which may be involved in intracellular signalling A human placental hbrary was extensively screened using three PTHrP ‘bait’ constructs Baits were ii&length PTHrP (aa l-141), mid-molecule (aa 38-94) and C-terminal (aa 107- 14 l), all of which have actions in different cellular systems Following several screens of the library, “prey” proteins interacting with only PTHrP (aa l-141) and (aa 107-141) were identified, and their interactions within the region aa 107-141 confirmed Two proteins were confirmed as positive within the system and indentified on the basis of sequence alignments. The first protein was identified as PA28 gamma, a modulator of proteasomat activity in the nucleus The second protein was identified as Betaarrestin, involved in G-coupled receptor desensitisation and internalisation The interaction of PTHrP with Beta-Arrestin or PA28 gamma could well be envisaged to affect the intracellular location of PTHrP, as well as modulating cellular activities

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