Ulrike Hügel

Dexamethasone Impairs Growth Hormone (GH)-Stimulated Growth by Suppression of Local Insulin-Like Growth Factor (IGF)-I Production and Expression of GH- and IGF-I-Receptor in Cultured Rat Chondrocytes1

Growth depression as a side effect of glucocorticoid therapy in childhood is partially mediated by alterations of the somatotropic hormone axis. The mechanisms of interaction between glucocorticoids and somatotropic hormones on the cellular and molecular level are poorly understood. In an experimental model of primary cultured rat growth plate chondrocytes, basal as well as GH (40 ng/ml) or insulin-like growth factor (IGF)-I (60 ng/ml)-stimulated growth was suppressed dose dependently (10−12–10−7 m) by dexamethasone (Dexa). An IGF-I antibody specifically and dose dependently inhibited the GH- but not the basic fibroblast growth factor (bFGF)-stimulated cell proliferation. GH increased the IGF-I concentration in conditioned serum-free culture medium; this was reversed by concomitant Dexa. Dexa time dependently suppressed the transcription of GH receptor (GHR) messenger RNA (mRNA) and down-regulated the basal and GH-stimulated expression of GHR. Whereas no suppressive effect on basal type I IGF-receptor (IG...

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat

SummaryVitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)2D3 receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [3H]-1,25(OH)2D3 is noted in both the logarithmic growth phase and at confluence (Nmax 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. KD was 10−11 M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)2D3 receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest Nmax in the proliferating cartilage. 1,25(OH)2D3 in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10−12 M and dose-dependent inhibition at 10−10 M and below. Inhibition was specific and not seen with 24,25(OH)2D3 or dexamethasone. Growth phase-dependent 1,25(OH)2D3 receptor expression and effects of 1,25(OH)2D3 on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.

Nuclear testicular 1,25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents.

1,25(OH)2D3 receptors were studied in whole testes, Sertoli cells, seminiferous tubules, Leydig cells and spermatogonia of adult NMRI mice and SD rats. Specific reversible high affinity binding (KD 1.4 x 10(-10)M; Nmax 72 fmol/mg protein) by a 3.5 S macromolecule was demonstrated in whole testes, Sertoli cells and seminiferous tubules. With identical techniques, no receptors were found in Leydig cells despite previous reports of 1,25(OH)2D3 actions on Leydig cell function.

Cultured Rat Growth Plate Chondrocytes Express Low Levels of 1α-Hydroxylase

Growth plate chondrocytes are the target of the hydroxylated vitamin D metabolites 1α,25(OH)2D3and 24,25(OH)2D3. Because studies on the production of these polar metabolites were inconclusive in various in vitro systems, the expression of a potential paracrine/autocrine vitamin D system was examined in primary cultures of rat growth plate chondrocytes using real-time RT-PCR. Compared to UMR cells and renal homogenates primary cultures of growth plate chondrocytes expressed low levels of 25-hydroxy-1a-hydroxylase as well as 25-hydroxy-24-hydroxylase. The expression of both is modulated by 25 vitamin D3, but 1α,25(OH)2D3affected only 25-hydroxy-24-hydroxylase. If these findings are confirmed in intact growth plates, the polar vitamin D metabolites could act in a paracrine/autocrine fashion within the growth plate.

Evidence for in vivo upregulation of 1,25(OH)2 vitamin D3 receptor in human monocytes.

SummaryMammalian cells increase net expression of 1,25(OH)2D3 receptors after exposure to physiological concentrations of 1,25(OH)2D3in vitro. we examined specific binding of 1,25(OH)2D3 by human monocytes before and after daily administration of 1.5–2 ug 1,25(OH)2D3 p.o. for 3 days in 5 healthy normal D-replete probands. Median specific binding (Nmax) at baseline was 793 molecules/cell and 2052 or 2828 at 24h and 72h of 1,25(OH)2D3 treatment respectively. The results suggest (a) up-regulation of 1,25(OH)2D3 receptors occurs in man and (b) monocyte preparations can be used to assess receptor regulationin vivo.

Rat growth plate chondrocytes express low levels of 25-hydroxy-1α-hydroxylase

Abstract Long standing disturbances of Vitamin D-metabolism as well as null-mutant animals for 25-hydroxy-1α-hydroxylase results in disorganised growth plates. Cultured chondrocytes were shown to be target for the hydroxylated Vitamin D-metabolites 1α,25(OH) 2 D 3 and 24,25(OH) 2 D 3 . Because studies on production of these metabolites were inconclusive in in vitro systems, the expression of the Vitamin D-system was examined in rat growth plate chondrocytes in vitro as well as ex vivo. Gene expression for 25-hydroxy-1α-hydroxylase, 25-hydroxy-24-hydroxylase as well as Vitamin D-receptor and collagen II and X were analysed on mRNA level by RT-PCR and quantitative real-time PCR, on protein level by western blotting and by immunohistochemistry in isolated growth plate chondrocytes or intact growth plates. Compared to UMR or CaCo 2 cells and renal homogenates cultured growth plate chondrocytes expressed low levels of 25-hydroxy-1α-hydroxylase mRNA and 25-hydroxy-24-hydroxylase mRNA. The expression of both was modulated by 25(OH)D 3 , but 1α,25(OH) 2 D 3 affected only 25-hydroxy-24-hydroxylase. These data were confirmed by Western blotting. Immunohistochemistry demonstrated predominant staining for 25-hydroxy-1α-hydroxylase in chondrocyte nodules and cells embedded in matrix in vitro. Ex vivo, 25-hydroxy-1α-hydroxylase was detected predominantly in late proliferative and hypertrophic zone of the growth plate. In conclusion, growth plate chondrocytes express the key components for a paracrine/autocrine Vitamin D-system.

Hyperparathyroidism and abnormal 1,25(OH)2vitamin D3 metabolism in experimental lead intoxication

Abstract. Clinical evidence points to disturbed calcium metabolism in lead (Pb) intoxication. To further clarify the mechanisms involved, serum levels of 1,25(OH)2D3, receptors for 1,25(OH)2D3 as well as size and ultrastructure of parathyroid glands were examined in Wistar Kyoto rats exposed to 1% lead (Pb) acetate in drinking water for 10 weeks (short‐term study) or 0·001 – 1 % Pb acetate for 24 weeks (long‐term study). After administration of Pb for 10 weeks, bone Pb was significantly increased (641±66·9 (SD) vs. 0·648±0·39 mg kg‐1 ash in controls). Total serum calcium and ionized Ca2+ (1·15±0·031 vs. 1·25±0·03 mmol 1‐1) were significantly decreased. Renal function (Ccr) was unchanged, but urinary cAMP excretion and circulating 1,25(OH)2D3 (177±10·9 vs. 232±18·9 pmol 1‐1) were diminished. Specific binding of 1,25(OH)2D3 was increased in parathyroids (Bmax 128±4·7 vs. 108±0·6 fmol mg‐1 protein) and intestinal mucosa; Bmax failed to adequately rise in response to pretreatment with 1,25(OH)2D3 (2·10 ng day‐1 for 4 d) in Pb‐exposed animals. Receptor characteristics (sedimentation constant, KD, DNA affinity) were unchanged. Parathyroid weight was significantly increased (178·25 vs. 96·34 μg) with no change of estimated nuclear volume, cell volume or cell ultrastructure. After 24 weeks of Pb exposure, a dose‐dependent but non‐linear increase of parathyroid weight was noted between 0·001% and 1% Pb in drinking fluid.